Genetically Modified Immune Cells Targeting NY-ESO-1 and Methods of Use Thereof

ABSTRACT

The present disclosure provides modified immune cell (e.g., modified T cell) comprising an exogenous T cell receptor (TCR) having specificity for NY-ESO-1. The present disclosure provides modified immune cells or precursors thereof (e.g., modified T cells) comprising an exogenous TCR and a switch receptor. Gene edited modified cells are also provided, such that the expression of one or more of an endogenous T-cell receptor gene (e.g., TRAC, TRBC) or an endogenous immune checkpoint gene (e.g. PD-1 or TIM-3) is downregulated.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/216,774, filed Dec. 11, 2018, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/597,717,filed Dec. 12, 2017, each of which is incorporated herein by referencein its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The Sequence Listing submitted herewith as a xml file named“046483-7193US2-Sequence-Listing,” created on Jul. 6, 2023 and having asize of 388,358 bytes, is incorporated herein by reference in itsentirety.

BACKGROUND

New York esophageal squamous cell carcinoma 1 (NY-ESO-1) protein isencoded by the CTAG1B gene, and was originally identified as a humantumor antigen by a method called serological expression (SEREX) cloningof recombinant cDNA libraries from human tumors. NY-ESO-1 belongs to thecancer-testis (CT) antigen group of proteins and its function remainsunknown.

Expression is restricted to the testis and ovaries as NY-ESO-1 has notbeen detected in other normal tissues by RT-PCR. On the other hand,NY-ESO-1 expression has been observed in various tumor types, includingmyelomas, sarcomas, melanomas and other solid tumors at both molecularand protein levels.

In multiple myeloma, detection of NY-ESO-1 varies depending on thestudy. Molecular and immunohistochemical analyses have demonstratedNY-ESO-1 expression ranges from approximately 7% to 56% of all multiplemyeloma samples tested. Overall, increased NY-ESO-1 has been observed inadvanced disease stages as well as myelomas with abnormal cytogeneticprofiles (approximately a third of the total patient population) acrossmultiple reports.

Expression of NY-ESO-1 protein in sarcoma samples has been reported tobe ≥80%. Synovial sarcoma (SS) and Myxoid/Round Cell Liposarcoma (MRCL)show the highest levels of expression. Results show strong andhomogeneous staining patterns, with more than 70% of SS and MRCL samplesdemonstrating intense signal in over half of the malignant cells. Insamples that are positive, NY-ESO-1 expression is intracellular andexpression is primarily cytoplasmic.

Melanoma samples have been shown to have varying degrees of NY-ESO-1expression depending on the study and metastatic status. One report hasshown that NY-ESO-1 is not expressed in primary tumors but detected in28% of metastatic samples, while two others demonstrated that ˜45% ofmelanoma samples were NY-ESO-1 positive, with no relationship betweenexpression and tumor stage. Furthermore, differential expression ofNY-ESO-1 has been observed in morphologically distinct metastases. Ithas also been suggested that NY-ESO-1 expression is positivelycorrelated with tumor thickness and negatively correlated withtumor-infiltrating lymphocytes.

Thus, there is a need in the art for novel tumor therapies targetingNY-ESO-1. The present invention addresses and satisfies this need.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that geneticallymodified immune cells (e.g., T cells) comprising an exogenous targetspecific TCR (e.g., NY-ESO-1 TCR) demonstrate enhanced efficacy inkilling target tumor cells in the tumor microenvironment. The immunecells may be genetically modified to comprise an exogenous receptor(e.g., a switch receptor) and/or may be genetically edited to disruptexpression of one or more endogenous receptors (e.g., a T-cell receptorgene and/or immune checkpoint protein) which suppress immune cellproliferation in the tumor microenvironment.

Accordingly, in certain aspects, the instant disclosure provides amodified T cell comprising an exogenous T cell receptor (TCR) havingaffinity for NY-ESO-1 on a target tumor cell, wherein the expression ofan endogenous receptor (e.g., a TCR alpha chain coding sequence, anendogenous TCR beta chain coding sequence, and/or an endogenous PD1coding sequence) is downregulated. In other aspects, the instantdisclosure provides a modified T cell comprising an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell and aswitch receptor, wherein the expression of an endogenous TCR alpha chaincoding sequence, and an endogenous TCR beta chain coding sequence isdownregulated.

In one aspect, a modified T cell comprising an exogenous T cell receptor(TCR) having affinity for NY-ESO-1 on a target cell, wherein theexpression of an endogenous TCR coding sequence and/or an endogenousimmune checkpoint coding sequence is downregulated, is provided.

In certain exemplary embodiments, the immune checkpoint coding sequenceencodes an immune checkpoint protein selected from the group consistingof PD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4(CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and VISTA.

In certain exemplary embodiments, the endogenous TCR coding sequence isTRAC or TRBC.

In certain exemplary embodiments, at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion (indel) is in theendogenous TCR alpha chain coding sequence comprising the nucleic acidsequence set forth in SEQ ID NO:128, thereby resulting in downregulatedexpression of the endogenous TCR alpha chain coding sequence.

In certain exemplary embodiments, at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion is in the endogenous TCRbeta chain coding sequence comprising the nucleic acid sequence setforth in SEQ ID NO:129, thereby resulting in downregulated expression ofthe endogenous TCR beta chain coding sequence.

In certain exemplary embodiments, at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion is in the endogenous PD1coding sequence comprising the nucleic acid sequence set forth in SEQ IDNO:130, thereby resulting in downregulated expression of the endogenousPD1 coding sequence.

In certain exemplary embodiments, optionally, at least one nucleotidesubstitution, deletion, insertion, and/or insertion/deletion is in theendogenous coding sequence selected from A2AR, B7-H3 (CD276), B7-H4(VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT,TIM-3, and VISTA, thereby resulting in downregulated expression of theendogenous A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96,CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and/or VISTA codingsequence.

In certain exemplary embodiments, the exogenous TCR comprises a TCRalpha chain comprising the amino acid sequence set forth in SEQ ID NO:5.

In certain exemplary embodiments, the exogenous TCR comprises a TCR betachain comprising the amino acid sequence set forth in SEQ ID NO:12.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; and c)at least one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129, whereinthe expression of the endogenous TCR alpha and beta chain codingsequences are downregulated, is provided.

In certain exemplary embodiments, the modified T cell further comprisesat least one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous coding sequence selected from PD1,A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152),IDO, KIR, LAG3, TIGIT, TIM-3, and VISTA.

In certain exemplary embodiments, the modified T cell further comprisesd) at least one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous PD1 coding sequence comprising thenucleic acid sequence set forth in SEQ ID NO:130.

In certain exemplary embodiments, the modified T cell is an autologous Tcell.

In certain exemplary embodiments, the autologous T cell is derived froma human.

In certain exemplary embodiments, the autologous T cell is a CD3+ Tcell.

In another aspect, a modified T cell comprising an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe expression of an endogenous TCR alpha chain coding sequence, and theexpression of an endogenous TCR beta chain coding sequence aredownregulated, is provided.

In certain exemplary embodiments, at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion is in the endogenous TCRalpha chain coding sequence comprising the nucleic acid sequence setforth in SEQ ID NO:128, thereby resulting in downregulated expression ofthe endogenous TCR alpha chain coding sequence.

In certain exemplary embodiments, at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion is in the endogenous TCRbeta chain coding sequence comprising the nucleic acid sequence setforth in SEQ ID NO:129, thereby resulting in downregulated expression ofthe endogenous TCR beta chain coding sequence.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; c) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129; and d)at least one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous PD1 coding sequence comprising thenucleic acid sequence set forth in SEQ ID NO:130; wherein the expressionof the endogenous TCR alpha chain, TCR beta chain coding sequence, andPD1 coding sequences are downregulated, is provided.

In certain exemplary embodiments, the modified T cell further comprisesat least one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous coding sequence selected from A2AR,B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO,KIR, LAG3, TIGIT, TIM-3, and VISTA.

In certain exemplary embodiments, the modified T cell further comprisesa switch receptor.

In certain exemplary embodiments, the switch receptor comprises: a firstdomain, wherein the first domain is derived from a first polypeptidethat is associated with a negative signal; and a second domain, whereinthe second domain is derived from a second polypeptide that isassociated with a positive signal.

In certain exemplary embodiments, the first domain comprises at least aportion of an extracellular domain of the first polypeptide that isassociated with a negative signal, and wherein the second domaincomprises at least a portion of an intracellular domain of the secondpolypeptide that is associated with a positive signal.

In certain exemplary embodiments, the switch receptor further comprisesa switch receptor transmembrane domain.

In certain exemplary embodiments, the switch receptor transmembranedomain comprises: a transmembrane domain of a first polypeptide that isassociated with a negative signal; or a transmembrane domain of a secondpolypeptide that is associated with a positive signal.

In certain exemplary embodiments, the first polypeptide that isassociated with a negative signal is selected from the group consistingof TIM-3, CTLA4, PD-1, BTLA, and TGFβR.

In certain exemplary embodiments, the first polypeptide that isassociated with a negative signal is a variant of PD-1 having analanine-to-leucine substitution at amino acid position 132 relative tothe wild-type PD-1 amino acid sequence.

In certain exemplary embodiments, the second polypeptide that isassociated with a positive signal is selected from the group consistingof 41BB, CD28, ICOS, and IL-12R. In certain exemplary embodiments, theswitch receptor comprises: a first domain comprising at least a portionof the extracellular domain of PD1; a switch receptor transmembranedomain comprising at least a portion of the transmembrane domain ofCD28; and a second domain comprising at least a portion of theintracellular domain of CD28.

In certain exemplary embodiments, the switch receptor comprises a firstdomain comprising at least a portion of the extracellular domain ofPD-1, wherein the PD-1 is a variant having an alanine-to-leucinesubstitution at amino acid position 132 relative to the wild-type PD-1amino acid sequence; a second domain comprising a switch receptortransmembrane domain comprising at least a portion of the transmembranedomain of CD28; and a third domain comprising at least a portion of theintracellular domain of CD28.

In certain exemplary embodiments, the switch receptor comprises a firstdomain comprising at least a portion of the extracellular domain ofPD-1; a second domain comprising a switch receptor transmembrane domaincomprising at least a portion of the transmembrane domain of CD8alpha;and a third domain comprising at least a portion of the intracellulardomain of 4-1BB.

In certain exemplary embodiments, the switch receptor comprises a firstdomain comprising at least a portion of the extracellular domain ofPD-1, wherein the PD-1 is a variant having an alanine-to-leucinesubstitution at amino acid position 132 relative to the wild-type PD-1amino acid sequence; a second domain comprising a switch receptortransmembrane domain comprising at least a portion of the transmembranedomain of CD8alpha; and a third domain comprising at least a portion ofthe intracellular domain of CD28.

In certain exemplary embodiments, the switch receptor comprises theamino acid sequence set forth in any one of SEQ ID NOs:14, 134, 136, or138.

In certain exemplary embodiments, the switch receptor comprises: a firstdomain comprising at least a portion of the extracellular domain ofTIM3; a switch receptor transmembrane domain comprising at least aportion of the transmembrane domain of CD28; and a second domaincomprising at least a portion of the intracellular domain of CD28.

In certain exemplary embodiments, the switch receptor comprises theamino acid sequence set forth in SEQ ID NO:132.

In certain exemplary embodiments, the switch receptor comprises: a firstdomain comprising at least a portion of the extracellular domain of aTGFβR; a second domain comprising at least a portion of theintracellular domain of a IL-12R.

In certain exemplary embodiments, the switch receptor comprises theamino acid sequence set forth in SEQ ID NO:16.

In certain exemplary embodiments, the switch receptor comprises theamino acid sequence set forth in SEQ ID NO:18.

In certain exemplary embodiments, the switch receptor comprises atruncated variant of a wild-type protein associated with a negativesignal.

In certain exemplary embodiments, the switch receptor comprises theamino acid sequence set forth in SEQ ID NO:20.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; c) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129; and d)a switch receptor comprising the amino acid sequence set forth in SEQ IDNO:14, wherein the expression of the endogenous TCR alpha chain codingsequence and the expression of the endogenous TCR beta chain codingsequence are downregulated, is provided.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; c) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129; and d)a switch receptor comprising the amino acid sequence set forth in SEQ IDNO:136, wherein the expression of the endogenous TCR alpha chain codingsequence and the expression of the endogenous TCR beta chain codingsequence are downregulated.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; c) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129; d) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TIM-3 coding sequence; and e) aswitch receptor comprising the amino acid sequence set forth in SEQ IDNO:14, wherein the expression of the endogenous TCR alpha chain codingsequence, the expression of the endogenous TCR beta chain codingsequence, and the expression of the endogenous TIM-3 coding sequence aredownregulated, is provided.

In another aspect, a modified T cell comprising: a) an exogenous T cellreceptor (TCR) having affinity for NY-ESO-1 on a target cell, whereinthe exogenous TCR comprises: (i) a TCR alpha chain comprising the aminoacid sequence set forth in SEQ ID NO:5; and (ii) a TCR beta chaincomprising the amino acid sequence set forth in SEQ ID NO:12; b) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR alpha chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:128; c) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129; d) atleast one nucleotide substitution, deletion, insertion, and/orinsertion/deletion in an endogenous coding sequence selected from A2AR,B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO,KIR, LAG3, PD1, TIGIT, TIM-3, and VISTA; and e) a switch receptorcomprising the amino acid sequence set forth in any one of SEQ IDNOs:14, 16, 18, 20, 132, 134, 136, and 138, wherein the expressions ofthe endogenous TCR alpha chain coding sequence, the endogenous TCR betachain coding sequence, and/or the endogenous coding sequence selectedfrom A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4(CD152), IDO, KIR, LAG3, PD1, TIGIT, TIM-3, or VISTA are downregulated,is provided.

In another aspect, a method for generating a modified T cell comprising:a) introducing into a T cell a first nucleic acid comprising a nucleicacid sequence encoding an exogenous T cell receptor (TCR) havingaffinity for NY-ESO-1 on a target cell; and b) introducing into the Tcell one or more nucleic acids capable of downregulating gene expressionof one or more endogenous genes selected from the group consisting ofTCR alpha chain, TCR beta chain, A2AR, B7-H3 (CD276), B7-H4 (VTCN1),BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, PD1, TIGIT, TIM-3,and VISTA, is provided.

In certain exemplary embodiments, the first nucleic acid comprises a TCRalpha chain coding sequence and a TCR beta chain coding sequence.

In certain exemplary embodiments, the TCR alpha chain coding sequenceand the TCR beta chain coding sequence are separated by a linker.

In certain exemplary embodiments, the linker comprises a nucleic acidsequence encoding an internal ribosome entry site (IRES).

In certain exemplary embodiments, the linker comprises a nucleic acidsequence encoding a self-cleaving peptide.

In certain exemplary embodiments, the self-cleaving peptide is a 2Apeptide.

In certain exemplary embodiments, the 2A peptide is selected from thegroup consisting of porcine teschovirus-1 2A (P2A), Thoseaasigna virus2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth diseasevirus 2A (F2A).

In certain exemplary embodiments, the 2A peptide is T2A.

In certain exemplary embodiments, the linker comprises a furin cleavagesite.

In certain exemplary embodiments, the linker comprises a furin cleavagesite and T2A.

In certain exemplary embodiments, the first nucleic acid comprises from5′ to 3′ the TCR alpha chain coding sequence, the linker, and the TCRbeta chain coding sequence.

In certain exemplary embodiments, the first nucleic acid comprises from5′ to 3′ the TCR beta chain coding sequence, the linker, and the TCRalpha chain coding sequence.

In certain exemplary embodiments, the TCR alpha chain coding sequencecomprises the nucleic acid sequence set forth in SEQ ID NO:6.

In certain exemplary embodiments, the TCR beta chain coding sequencecomprises the nucleic acid sequence set forth in SEQ ID NO:13.

In certain exemplary embodiments, the first nucleic acid is introducedby viral transduction.

In certain exemplary embodiments, the viral transduction comprisescontacting the cell with a viral vector comprising the first nucleicacid.

In certain exemplary embodiments, the viral vector is selected from thegroup consisting of a retroviral vector, a lentiviral vector, anadenoviral vector, and an adeno-associated viral vector.

In certain exemplary embodiments, the viral vector is a lentiviralvector.

In certain exemplary embodiments, the lentiviral vector furthercomprises an EF-1α promoter.

In certain exemplary embodiments, the lentiviral vector furthercomprises a rev response element (RRE).

In certain exemplary embodiments, the lentiviral vector furthercomprises a woodchuck hepatitis virus posttranscriptional regulatoryelement (WPRE).

In certain exemplary embodiments, the lentiviral vector furthercomprises a cPPT sequence.

In certain exemplary embodiments, the lentiviral vector furthercomprises an EF-1α promoter, a rev response element (RRE), a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE), and acPPT sequence.

In certain exemplary embodiments, the lentiviral vector is aself-inactivating lentiviral vector.

In certain exemplary embodiments, each of the one or more nucleic acidscapable of downregulating gene expression comprises an antisense RNA, anantagomir RNA, siRNA, shRNA, and a CRISPR system, or any combinationthereof.

In certain exemplary embodiments, the CRISPR system comprises a Cas9 RNAand a guide RNA (gRNA).

In certain exemplary embodiments, the CRISPR system comprises aCas9/gRNA ribonucleoprotein complex.

In certain exemplary embodiments, the CRISPR system comprises a gRNAcomprising a nucleic acid sequence set forth in any one of SEQ IDNOs:37-127 and 131.

In certain exemplary embodiments, each of the one or more nucleic acidscapable of downregulating gene expression is introduced byelectroporation.

In another aspect, a method for generating a modified T cell comprising:a) introducing into a T cell a first nucleic acid comprising a nucleicacid sequence encoding an exogenous T cell receptor (TCR) havingaffinity for NY-ESO-1 on a target cell; and b) introducing into the Tcell one or more nucleic acids capable of downregulating gene expressionof one or more endogenous genes selected from the group consisting ofTCR alpha chain, and TCR beta chain, is provided.

In certain exemplary embodiments, the first nucleic acid furthercomprises a nucleic acid sequence encoding a switch receptor.

In certain exemplary embodiments, the nucleic acid sequence encoding theswitch receptor comprises the nucleic acid sequence set forth in SEQ IDNO:15.

In certain exemplary embodiments, the nucleic acid sequence encoding theswitch receptor comprises the nucleic acid sequence set forth in SEQ IDNO: 137.

In certain exemplary embodiments, the nucleic acid sequence encoding theswitch receptor comprises the nucleic acid sequence set forth in SEQ IDNO: 139.

In certain exemplary embodiments, the method further comprises: c)introducing into the T cell one or more nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom the group consisting A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA(CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and VISTA.

In another aspect, a method for generating a modified T cell comprising:a) introducing into a T cell a first nucleic acid comprising a nucleicacid sequence encoding an exogenous T cell receptor (TCR) havingaffinity for NY-ESO-1 on a target cell, and a nucleic acid sequenceencoding a switch receptor; and b) introducing into the T cell one ormore nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of TCR alphachain, TCR beta chain, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272),CD96, CTLA-4 (CD152), IDO, KIR, LAG3, PD1, TIGIT, TIM-3, and VISTA, isprovided.

In certain exemplary embodiments, the nucleic acid sequence encoding theexogenous TCR comprises a TCR alpha chain coding sequence and a TCR betachain coding sequence.

In certain exemplary embodiments, the TCR alpha chain coding sequencecomprises the nucleic acid sequence set forth in SEQ ID NO:6.

In certain exemplary embodiments, the TCR beta chain coding sequencecomprises the nucleic acid sequence set forth in SEQ ID NO:13.

In certain exemplary embodiments, the nucleic acid sequence encoding theswitch receptor comprises the nucleic acid sequence set forth in SEQ IDNO:15, 17, 19, or 21, 133, 135, 137 or 139.

In certain exemplary embodiments, the nucleic acid sequence encoding theexogenous TCR and the nucleic acid sequence encoding the switch receptorare separated by a first linker.

In certain exemplary embodiments, the TCR alpha chain coding sequenceand the TCR beta chain coding sequence are separated by a second linker.

In certain exemplary embodiments, each of the first and second linkersindependently comprise a nucleic acid sequence encoding an internalribosome entry site (IRES).

In certain exemplary embodiments, each of the first and second linkersindependently comprise a nucleic acid sequence encoding a self-cleavingpeptide.

In certain exemplary embodiments, the self-cleaving peptide is a 2Apeptide.

In certain exemplary embodiments, the 2A peptide is selected from thegroup consisting of porcine teschovirus-1 2A (P2A), Thoseaasigna virus2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth diseasevirus 2A (F2A).

In certain exemplary embodiments, the 2A peptide is T2A.

In certain exemplary embodiments, the first linker and the second linkerare different.

In certain exemplary embodiments, the second linker comprises a nucleicacid sequence encoding a furin cleavage site.

In certain exemplary embodiments, the second linker comprises a nucleicacid sequence encoding a furin cleavage site and T2A.

In certain exemplary embodiments, the first linker comprises a nucleicacid sequence encoding F2A.

In certain exemplary embodiments, the first nucleic acid comprises from5′ to 3′ the nucleic acid sequence encoding the switch receptor, thefirst linker, the TCR alpha chain coding sequence, the second linker,and the TCR beta chain coding sequence.

In certain exemplary embodiments, the first nucleic acid comprises from5′ to 3′ the TCR beta chain coding sequence, the second linker, the TCRalpha chain coding sequence, the first linker, and the nucleic acidsequence encoding the switch receptor.

In certain exemplary embodiments, the first nucleic acid is introducedby viral transduction.

In certain exemplary embodiments, the viral transduction comprisescontacting the cell with a viral vector comprising the first nucleicacid.

In certain exemplary embodiments, the viral vector is selected from thegroup consisting of a retroviral vector, a lentiviral vector, anadenoviral vector, and an adeno-associated viral vector.

In certain exemplary embodiments, the viral vector is a lentiviralvector.

In certain exemplary embodiments, the lentiviral vector furthercomprises an EF-1α promoter.

In certain exemplary embodiments, the lentiviral vector furthercomprises a rev response element (RRE).

In certain exemplary embodiments, the lentiviral vector furthercomprises a woodchuck hepatitis virus posttranscriptional regulatoryelement (WPRE).

In certain exemplary embodiments, the lentiviral vector furthercomprises a cPPT sequence.

In certain exemplary embodiments, the lentiviral vector furthercomprises an EF-1α promoter, a rev response element (RRE), a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE), and acPPT sequence.

In certain exemplary embodiments, the lentiviral vector is aself-inactivating lentiviral vector.

In certain exemplary embodiments, each of the one or more nucleic acidscapable of downregulating gene expression comprises an antisense RNA, anantagomir RNA, siRNA, shRNA, and a CRISPR system, or any combinationthereof.

In certain exemplary embodiments, the CRISPR system is a Cas9 RNA and agRNA.

In certain exemplary embodiments, the CRISPR system is a Cas9/gRNA RNPcomplex.

In certain exemplary embodiments, the CRISPR system comprises a gRNAcomprising a nucleic acid sequence set forth in any one of SEQ ID NOs:37-127, and 131.

In certain exemplary embodiments, each of the one or more nucleic acidscapable of downregulating gene expression is introduced byelectroporation.

In another aspect, a method of treating cancer in a subject in needthereof, the method comprising administering a therapeutically effectivecomposition comprising a modified immune cell described herein to thesubject, is provided.

In another aspect, a method of treating multiple myeloma in a subject inneed thereof, comprising: a) administering to the subject alymphodepleting chemotherapy comprising administering to the subject aneffective amount of cyclophosphamide; b) administering to the subject amodified T cell comprising: i) an exogenous T cell receptor (TCR) havingaffinity for NY-ESO-1 on a target cell, wherein the exogenous TCRcomprises: (1) a TCR alpha chain comprising the amino acid sequence setforth in SEQ ID NO:5; and (2) a TCR beta chain comprising the amino acidsequence set forth in SEQ ID NO:12; ii) at least one nucleotidesubstitution, deletion, insertion, and/or insertion/deletion in anendogenous TCR alpha chain coding sequence comprising the nucleic acidsequence set forth in SEQ ID NO:128; iii) at least one nucleotidesubstitution, deletion, insertion, and/or insertion/deletion in anendogenous TCR beta chain coding sequence comprising the nucleic acidsequence set forth in SEQ ID NO:129; and iv) at least one nucleotidesubstitution, deletion, insertion, and/or insertion/deletion in anendogenous PD1 coding sequence comprising the nucleic acid sequence setforth in SEQ ID NO:130, wherein the expression of the endogenous TCRalpha chain coding sequence, the endogenous TCR beta chain codingsequence, and endogenous PD1 coding sequence are downregulated, isprovided.

In another aspect, a method of treating melanoma, synovial sarcoma, ormyxoid/round cell liposarcoma in a subject in need thereof, comprising:a) administering to the subject a lymphodepleting chemotherapycomprising administering to the subject an effective amount ofcyclophosphamide, and an effective amount of fludarabine; b)administering to the subject a modified T cell comprising: i) anexogenous T cell receptor (TCR) having affinity for NY-ESO-1 on a targetcell, wherein the exogenous TCR comprises: (1) a TCR alpha chaincomprising the amino acid sequence set forth in SEQ ID NO:5; and (2) aTCR beta chain comprising the amino acid sequence set forth in SEQ IDNO:12; ii) at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion in an endogenous TCR alpha chain codingsequence comprising the nucleic acid sequence set forth in SEQ IDNO:128; iii) at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion in an endogenous TCR beta chain codingsequence comprising the nucleic acid sequence set forth in SEQ IDNO:129; and iv) at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in an endogenous PD1 codingsequence comprising the nucleic acid sequence set forth in SEQ IDNO:130, wherein the expression of the endogenous TCR alpha chain codingsequence, the endogenous TCR beta chain coding sequence, and endogenousPD1 coding sequence are downregulated, is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIG. 1 depicts sequencing of a portion of the endogenous TRAC gene incell clones targeted by the Group I TRAC gRNA.

FIG. 2 depicts sequencing of a portion of the endogenous TRBC gene incell clones targeted by the Group I TRBC gRNA.

FIG. 3 depicts sequencing of a portion of the endogenous PDCD1 gene incell clones targeted by the Group I PD1 gRNA.

FIG. 4 depicts graphs demonstrating the expansion of NY-ESO-1 TCRtransduced T cells with different concentrations of Cas9 RNA plus gRNAsfor TCRα, β, and PD1.

FIG. 5 depicts flow cytometry analysis demonstrating the efficiency ofgene editing of endogenous TCR.

FIG. 6 depicts a graph demonstrating that surface expression of theNY-ESO-1 specific TCR is enhanced by increasing doses of endogenousTCR-targeting gRNAs.

FIG. 7 depicts flow cytometry analysis demonstrating the efficiency ofPD1 gene editing.

FIG. 8 depicts results from a Surveyor assay for detection of percentgene editing.

FIG. 9 depicts flow cytometry analysis demonstrating the ability ofNY-ESO-1 transduced T cell with TCR^(endo) and PD1 gene edited todegranulate in response to tumor.

FIG. 10 depicts graphs demonstrating the ability of NY-ESO-1 transducedT cell with TCR^(endo) and PD1 gene editing to release IFNγ or IL-2 inresponse to tumor.

FIG. 11 depicts a graph demonstrating the ability of NY-ESO-1 transducedT cell with TCR^(endo) and PD1 gene editing to lyse NY-ESO-1 expressingtumor.

FIG. 12 depicts graphs demonstrating the long term culture of NY-ESO-1transduced T cells with TCR^(endo) and PD1 gene editing.

FIG. 13 depicts a graph showing NY-ESO-1 TCR expression at the end oflong term cultures.

FIG. 14 depicts a schema describing a general protocol for theadministration of NYCE T cells.

FIGS. 15A-15C depict the characterization of T cells transferred with 8FNY-ESO-1 TCR via electroporation.

FIGS. 16A-16B depict results of stress tests that were performed onCRISPR gene edited NY-ESO-1 TCR transduced T cells.

FIG. 17 depicts graphs showing the level of cytokine production instimulated gene edited T cells.

FIG. 18 depicts flow cytometry analysis of 8F, 1G4, or Ly95 transduced Tcells.

FIG. 19 depicts the level of cytokine production of differentNY-ESO-1/HLA-A2 positive cell lines.

FIG. 20 depicts the lytic activity of different TRAC and TRBC disruptedT cells.

FIGS. 21A-21B depict data showing that disruption of TRAC and TRBCimproves function of NY-ESO-1 TCR transduced T cells.

FIG. 22 depicts a lentiviral map comprising an NY-ESO-1 TCR and PD1-CD28switch receptor.

FIGS. 23A-23B depict data showing the in vitro function tests ofCRISPR/CAS9 gene edited, NY-ESO-1 TCR (8F) transduced T cells, with orwithout co-expressing the PD1-CD28 switch receptor.

FIGS. 24A-24B depict bioluminescence imaging and data for a first set ofmouse experiments.

FIGS. 25A-25D depict bioluminescence imaging and data for a second setof mouse experiments. FIG. 25D shows a graph of tumor size that wasmeasured one day prior to the T cell treatment, and weeklypost-treatment.

FIGS. 26A-26C depict a series of plots illustrating CRISPR/Cas9 genedisruption efficiency of T cells.

FIGS. 27A-27D depict the off-target detection of gRNAs using Guide-seq.

FIGS. 28A-28D depict a series of plots illustrating CRISPR/Cas9 genedisruption efficiency of T cells.

FIGS. 29A-29B depict data showing further efficacy in disrupting TIM-3.

FIGS. 30A-30C depict data showing improved anti-tumor function ofNY-ESO-1 TCR transduced T cells with PD1-CD28 switch receptor.

FIGS. 31A-31F depict data showing massive gene expression profilechanges in transduced T cells.

FIGS. 31G-31H depict data showing gene expression distribution andprofile changes in various T cells as indicated.

FIGS. 32A-32B depict data showing that T cells transduced with NY-ESO-1TCR (8F) and PD1-CD28 switch receptor exhibit increased resistance toTGFβ and adenosine.

FIGS. 33A-33B depict data showing that T cells transduced with NY-ESO-1TCR (8F) and PD1-CD28 switch receptor exhibit increased resistanceTregs.

FIG. 33C depicts data demonstrating enhanced resistance of variousNY-ESO-1 TCR (8F) T cells as indicated, to hypoxia inhibition.

FIGS. 34A-34B depict data showing that a TGFβR-IL12R switch receptorimproves NY-ESO-1 TCR transduced T cell function.

FIGS. 35A-35C depict data showing that a high affinity PD1 switchreceptor enhances NY-ESO-1 TCR anti-tumor activity. Bioluminescenceimages are shown in FIG. 35A, and corresponding quantification ofradiance is shown in FIG. 35B. FIG. 35C is a plot showing change intumor size over time for the various groups as indicated. UTD:untransduced; CD28: NY-ESO-1 TCR (8F) and PD1.CD28 switch (PD1-CD28);CD28 #: NY-ESO-1 TCR (8F) and PD1*.CD28 switch (PD1^(A132L)-CD28); BB:NY-ESO-1 TCR (8F) and PD1.BB switch (PD1-41BB); BB #: NY-ESO-1 TCR (8F)and PD1*.BB switch (PD1^(A132L)-41BB); 8F: NY-ESO-1 TCR.

FIGS. 36A-36E depict data showing that a high affinity PD1 switchreceptor enhances NY-ESO-1 TCR anti-tumor activity. FIG. 36A showsbioluminescence images, and corresponding quantification of radiance isshown in FIG. 36B. FIG. 36C is a plot showing change in tumor size overtime for the various groups as indicated. A: untransduced, TRAC/TRBCdisrupted T cells (UTD DKO); B: NY-ESO-1 TCR (8F), TRAC/TRBC disrupted Tcells (8F DKO); C: NY-ESO-1 TCR (8F), TRAC/TRBC/PDCD1/TIM3 disrupted Tcells (8F DKO+PD1 & Tim3 KO); D: NY-ESO-1 TCR (8F), TIM3-CD28 switch,TRAC/TRBC disrupted T cells (Tim3CD28.8F DKO); E: NY-ESO-1 TCR (8F),TIM3-CD28 switch, TRAC/TRBC/PDCD1 disrupted T cells (Tim3CD28.8F DKO+PD1KO); F: NY-ESO-1 TCR (8F), PD1^(A132L)-41BB switch, TRAC/TRBC disruptedT cells (PD1*BB.8F DKO); G: NY-ESO-1 TCR (8F), PD1^(A132L)-41BB switch,TRAC/TRBC/TIM3 disrupted T cells (PD1*BB.8F DKO+ Tim3 KO); H: NY-ESO-1TCR (8F), PD1^(A132L)-41BB switch, TIM3-CD28 switch, TRAC/TRBC disruptedT cells (PD1*BB.Tim3CD28.8F DKO); and I: NY-ESO-1 TCR (8F), PD1-CD28switch, TRAC/TRBC/TIM3 disrupted T cells (PD1CD28.8F DKO+ Tim3 KO). FIG.36D shows BLI of mice injected with T cells as indicated. FIG. 36E showsa plot of tumor size measured at various time points as indicatedpost-injection with T cells for the various groups as indicated. InFIGS. 36D and 36E: UTD: untransduced T cells; PD1.CD28.8F (CD28 in FIG.36E): 8F NY-ESO-1 TCR T cells with PD1-CD28 switch; PD1*.CD28.8F (CD28#in FIG. 36E): 8F NY-ESO-1 TCR T cells with PD1^(A132L)-CD28 switch;PD1.BB.8F (BB in FIG. 36E): 8F NY-ESO-1 TCR T cells with PD1-41 BBswitch; PD1*.BB.8F (BB #in FIG. 36E): 8F NY-ESO-1 TCR T cells withPD1^(A132L)-41BB switch; 8F: 8F NY-ESO-1 TCR T cells.

DETAILED DESCRIPTION

The present invention provides compositions and methods for modifiedimmune cells (e.g., T cells and NK cells) or precursors thereof (e.g.,modified T cells) comprising an exogenous (e.g., recombinant, transgenicor engineered) T cell receptor (TCR). In some embodiments, the exogenousTCR is a TCR having affinity for NY-ESO-1 on a target cell (NY-ESO-1TCR), particularly melanoma and other solid tumor cells. The providedcells comprise additional genetic modifications to enhance the efficacyof the immune cell in the tumor microenvironment. In certainembodiments, the immune cells have a genetic disruption of a geneencoding endogenous TCR polypeptide (e.g., TRAC or TRBC) or anendogenous immune checkpoint protein (e.g., PD1 or TIM3). In additionalor alternative embodiments, the immune cell comprises an exogenousswitch molecule. In some embodiments, the modified immune cell orprecursor thereof (e.g., modified T cell) further comprises a switchreceptor. In some embodiments, the modified immune cell or precursorthereof (e.g., modified T cell comprising an NY-ESO-1 TCR) is furthermodified such that the expression of one or more of TRAC and/or TRBC isdownregulated. In additional or alternative embodiments, the modifiedimmune cell is further modified such that expression of an immunecheckpoint protein (e.g., PD1 or TIM3) is downregulated. In oneembodiment, the present invention provides a modified T cell comprisingan exogenous TCR having affinity for NY-ESO-1 on a target cell, whereinthe T cell is further modified such that the expression of TRAC, TRBC,and PD-1 is downregulated. In another embodiment, the present inventionprovides a modified T cell comprising an exogenous TCR having affinityfor NY-ESO-1 on a target cell, and a switch receptor, wherein the T cellis further modified such that the expression of TRAC and TRBC isdownregulated. Also provided are methods of producing such geneticallyengineered cells. In some embodiments, the cells and compositions can beused in adoptive cell therapy, e.g. adoptive tumor immunotherapy.

In some embodiments, the provided immune cells, compositions and methodsalter or reduce the effects of T cell inhibitory pathways or signals inthe tumor microenvironment. The modified immune cells of the inventioncounteract the upregulation and/or expression of inhibitory receptor orligands that can negatively control T cell activation and T cellfunction. For example, expression of certain immune checkpoint proteins(e.g., PD-1 or PD-LI) on T cells and/or in the tumor microenvironmentcan reduce the potency and efficacy of adoptive T cell therapy. Suchinhibitory pathways may otherwise impair certain desirable effectorfunctions in the context of adoptive cell therapy. Tumor cells and/orcells in the tumor microenvironment often upregulate certain inhibitoryproteins (such as PD-L1 and PD-L2) delivering an inhibitory signal. Suchproteins may also be upregulated on T cells in the tumormicroenvironment, e.g., on tumor-infiltrating T cells, which can occurfollowing signaling through the antigen receptor or certain otheractivating signals. Such events may contribute to genetically engineeredimmune cells (e.g., NY-ESO-1 targeting T cells) acquiring an exhaustedphenotype, such as when present in proximity with other cells thatexpress such protein, which in turn can lead to reduced functionality.Thus, the modified immune cells of the invention address the T cellexhaustion and/or the lack of T cell persistence that is a barrier tothe efficacy and therapeutic outcomes of conventional adoptive celltherapies.

It is to be understood that the methods described in this disclosure arenot limited to particular methods and experimental conditions disclosedherein as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements, Molecular Cloning: A Laboratory Manual(Fourth Edition) by M R Green and J. Sambrook and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (2013, 2nd edition).

A. Definitions

Unless otherwise defined, scientific and technical terms used hereinhave the meanings that are commonly understood by those of ordinaryskill in the art. In the event of any latent ambiguity, definitionsprovided herein take precedent over any dictionary or extrinsicdefinition. Unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein is well-knownand commonly used in the art. The methods and techniques provided hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

That the disclosure may be more readily understood, select terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or +10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including virtually all proteins or peptides, can serveas an antigen.

Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequence or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to elicit the desired immune response. Moreover,a skilled artisan will understand that an antigen need not be encoded bya “gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “downregulation” as used herein refers to the decrease orelimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited toan amount that when administered to a mammal, causes a detectable levelof immune suppression or tolerance compared to the immune responsedetected in the absence of the composition of the invention. The immuneresponse can be readily assessed by a plethora of art-recognizedmethods. The skilled artisan would understand that the amount of thecomposition administered herein varies and can be readily determinedbased on a number of factors such as the disease or condition beingtreated, the age and health and physical condition of the mammal beingtreated, the severity of the disease, the particular compound beingadministered, and the like.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly about 10 amino acids and/or sugars in size.Preferably, the epitope is about 4-18 amino acids, more preferably about5-16 amino acids, and even more most preferably 6-14 amino acids, morepreferably about 7-12, and most preferably about 8-10 amino acids. Oneskilled in the art understands that generally the overallthree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity andtherefore distinguishes one epitope from another. Based on the presentdisclosure, a peptide of the present invention can be an epitope.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anarginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

The term “immunosuppressive” is used herein to refer to reducing overallimmune response.

“Insertion/deletion”, commonly abbreviated “indel,” is a type of geneticpolymorphism in which a specific nucleotide sequence is present(insertion) or absent (deletion) in a genome.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “knockdown” as used herein refers to a decrease in geneexpression of one or more genes.

The term “knockout” as used herein refers to the ablation of geneexpression of one or more genes.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

A “target site” or “target sequence” refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (α) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver. A transplant can alsorefer to any material that is to be administered to a host. For example,a transplant can refer to a nucleic acid or a protein.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to, Sendaiviral vectors, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

B. T Cell Receptors

The present invention provides compositions and methods for modifiedimmune cells or precursors thereof (e.g., modified T cells) comprisingan exogenous T cell receptor (TCR). Thus, in some embodiments, thetarget cell has been altered to contain specific T cell receptor (TCR)genes (e.g., a TRAC and TRBC gene). TCRs or antigen-binding portionsthereof include those that recognize a peptide epitope or T cell epitopeof a target polypeptide, such as an antigen of a tumor, viral orautoimmune protein. In some embodiments, the TCR has binding specificityfor a tumor associated antigen, e.g., human NY-ESO-1.

A TCR is a disulfide-linked heterodimeric protein comprised of sixdifferent membrane bound chains that participate in the activation of Tcells in response to an antigen. There exists alpha/beta TCRs andgamma/delta TCRs. An alpha/beta TCR comprises a TCR alpha chain and aTCR beta chain. T cells expressing a TCR comprising a TCR alpha chainand a TCR beta chain are commonly referred to as alpha/beta T cells.Gamma/delta TCRs comprise a TCR gamma chain and a TCR delta chain. Tcells expressing a TCR comprising a TCR gamma chain and a TCR deltachain are commonly referred to as gamma/delta T cells. A TCR of thepresent disclosure is a TCR comprising a TCR alpha chain and a TCR betachain.

The TCR alpha chain and the TCR beta chain are each comprised of twoextracellular domains, a variable region and a constant region. The TCRalpha chain variable region and the TCR beta chain variable region arerequired for the affinity of a TCR to a target antigen. Each variableregion comprises three hypervariable or complementarity-determiningregions (CDRs) which provide for binding to a target antigen. Theconstant region of the TCR alpha chain and the constant region of theTCR beta chain are proximal to the cell membrane. A TCR furthercomprises a transmembrane region and a short cytoplasmic tail. CD3molecules are assembled together with the TCR heterodimer. CD3 moleculescomprise a characteristic sequence motif for tyrosine phosphorylation,known as immunoreceptor tyrosine-based activation motifs (ITAMs).Proximal signaling events are mediated through the CD3 molecules, andaccordingly, TCR-CD3 complex interaction plays an important role inmediating cell recognition events.

Stimulation of TCR is triggered by major histocompatibility complexmolecules (MHCs) on antigen presenting cells that present antigenpeptides to T cells and interact with TCRs to induce a series ofintracellular signaling cascades. Engagement of the TCR initiates bothpositive and negative signaling cascades that result in cellularproliferation, cytokine production, and/or activation-induced celldeath.

A TCR of the present invention can be a wild-type TCR, a high affinityTCR, and/or a chimeric TCR. A high affinity TCR may be the result ofmodifications to a wild-type TCR that confers a higher affinity for atarget antigen compared to the wild-type TCR. A high affinity TCR may bean affinity-matured TCR. Methods for modifying TCRs and/or theaffinity-maturation of TCRs are known to those of skill in the art.Techniques for engineering and expressing TCRs include, but are notlimited to, the production of TCR heterodimers which include the nativedisulphide bridge which connects the respective subunits (Garboczi, etal., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), JImmunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91:11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21):15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S.Pat. No. 6,080,840).

In some embodiments, the exogenous TCR is a full TCR or antigen-bindingportions or antigen-binding fragments thereof. In some embodiments, theTCR is an intact or full-length TCR, including TCRs in the αβ form or γδform. In some embodiments, the TCR is an antigen-binding portion that isless than a full-length TCR but that binds to a specific peptide boundin an MHC molecule, such as binds to an MHC-peptide complex. In somecases, an antigen-binding portion or fragment of a TCR can contain onlya portion of the structural domains of a full-length or intact TCR, butyet is able to bind the peptide epitope, such as MHC-peptide complex, towhich the full TCR binds. In some cases, an antigen-binding portioncontains the variable domains of a TCR, such as variable a chain andvariable β chain of a TCR, sufficient to form a binding site for bindingto a specific MHC-peptide complex. Generally, the variable chains of aTCR contain complementarity determining regions (CDRs) involved inrecognition of the peptide, MHC and/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or CDRs, which generally are the primarycontributors to antigen recognition and binding capabilities andspecificity. In some embodiments, a CDR of a TCR or combination thereofforms all or substantially all of the antigen-binding site of a givenTCR molecule. The various CDRs within a variable region of a TCR chaingenerally are separated by framework regions (FRs), which generallydisplay less variability among TCR molecules as compared to the CDRs(see, e.g., Jores et al, Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990;Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev.Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDRresponsible for antigen binding or specificity, or is the most importantamong the three CDRs on a given TCR variable region for antigenrecognition, and/or for interaction with the processed peptide portionof the peptide-MHC complex. In some contexts, the CDR1 of the alphachain can interact with the N-terminal part of certain antigenicpeptides. In some contexts, CDR1 of the beta chain can interact with theC-terminal part of the peptide. In some contexts, CDR2 contributes moststrongly to or is the primary CDR responsible for the interaction withor recognition of the MHC portion of the MHC-peptide complex. In someembodiments, the variable region of the β-chain can contain a furtherhypervariable region (CDR4 or HVR4), which generally is involved insuperantigen binding and not antigen recognition (Kotb (1995) ClinicalMicrobiology Reviews, 8:411-426).

In some embodiments, a TCR contains a variable alpha domain (V_(a))and/or a variable beta domain (V_(β)) or antigen-binding fragmentsthereof. In some embodiments, the a-chain and/or β-chain of a TCR alsocan contain a constant domain, a transmembrane domain and/or a shortcytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The ImmuneSystem in Health and Disease, 3 Ed., Current Biology Publications, p.4:33, 1997). In some embodiments, the a chain constant domain is encodedby the TRAC gene (IMGT nomenclature) or is a variant thereof. In someembodiments, the β chain constant region is encoded by TRBC1 or TRBC2genes (IMGT nomenclature) or is a variant thereof. In some embodiments,the constant domain is adjacent to the cell membrane. For example, insome cases, the extracellular portion of the TCR formed by the twochains contains two membrane-proximal constant domains, and twomembrane-distal variable domains, which variable domains each containCDRs.

It is within the level of a skilled artisan to determine or identify thevarious domains or regions of a TCR. In some aspects, residues of a TCRare known or can be identified according to the InternationalImmunogenetics Information System (IMGT) numbering system (see e.g.www.imgt.org; see also, Lefranc et al. (2003) Developmental andComparative Immunology, 2&; 55-77; and The T Cell Factsbook 2nd Edition,Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1sequences within a TCR Va chain and/or vβ chain correspond to the aminoacids present between residue numbers 27-38, inclusive, the CDR2sequences within a TCR Va chain and/or vβ chain correspond to the aminoacids present between residue numbers 56-65, inclusive, and the CDR3sequences within a TCR Va chain and/or vβ chain correspond to the aminoacids present between residue numbers 105-117, inclusive.

In some embodiments, the TCR may be a heterodimer of two chains a and β(or optionally γ and δ) that are linked, such as by a disulfide bond ordisulfide bonds. In some embodiments, the constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe a and β chains, such that the TCR contains two disulfide bonds inthe constant domains. In some embodiments, each of the constant andvariable domains contain disulfide bonds formed by cysteine residues.

In some embodiments, the TCR for engineering cells as described is onegenerated from a known TCR sequence(s), such as sequences of vα,βchains, for which a substantially full-length coding sequence is readilyavailable. Methods for obtaining full-length TCR sequences, including Vchain sequences, from cell sources are well known. In some embodiments,nucleic acids encoding the TCR can be obtained from a variety ofsources, such as by polymerase chain reaction (PCR) amplification ofTCR-encoding nucleic acids within or isolated from a given cell orcells, or synthesis of publicly available TCR DNA sequences. In someembodiments, the TCR is obtained from a biological source, such as fromcells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomasor other publicly available source. In some embodiments, the T-cells canbe obtained from in vivo isolated cells. In some embodiments, theT-cells can be a cultured T-cell hybridoma or clone. In someembodiments, the TCR or antigen-binding portion thereof can besynthetically generated from knowledge of the sequence of the TCR. Insome embodiments, a high-affinity T cell clone for a target antigen(e.g., a cancer antigen) is identified, isolated from a patient, andintroduced into the cells. In some embodiments, the TCR clone for atarget antigen has been generated in transgenic mice engineered withhuman immune system genes (e.g., the human leukocyte antigen system, orHLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) ClinCancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol.175:5799-5808. In some embodiments, phage display is used to isolateTCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008)Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al. (2003) NatImmunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al. (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments as described, the TCR can contain an introduceddisulfide bond or bonds. In some embodiments, the native disulfide bondsare not present. In some embodiments, the one or more of the nativecysteines (e.g. in the constant domain of the a chain and β chain) thatform a native interchain disulfide bond are substituted to anotherresidue, such as to a serine or alanine. In some embodiments, anintroduced disulfide bond can be formed by mutating non-cysteineresidues on the alpha and beta chains, such as in the constant domain ofthe a chain and β chain, to cysteine. Exemplary non-native disulfidebonds of a TCR are described in published International PCT No.WO2006/000830 and WO2006037960. In some embodiments, cysteines can beintroduced at residue Thr48 of the a chain and Ser57 of the β chain, atresidue Thr45 of the a chain and Ser77 of the β chain, at residue TyrIOof the a chain and SerI7 of the β chain, at residue Thr45 of the a chainand Asp59 of the β chain and/or at residue SerI5 of the a chain andGluI5 of the β chain. In some embodiments, the presence of non-nativecysteine residues (e.g. resulting in one or more non-native disulfidebonds) in a recombinant TCR can favor production of the desiredrecombinant TCR in a cell in which it is introduced over expression of amismatched TCR pair containing a native TCR chain.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some aspects,each chain (e.g. alpha or beta) of the TCR can possess one N-terminalimmunoglobulin variable domain, one immunoglobulin constant domain, atransmembrane region, and a short cytoplasmic tail at the C-terminalend. In some embodiments, a TCR, for example via the cytoplasmic tail,is associated with invariant proteins of the CD3 complex involved inmediating signal transduction. In some cases, the structure allows theTCR to associate with other molecules like CD3 and subunits thereof. Forexample, a TCR containing constant domains with a transmembrane regionmay anchor the protein in the cell membrane and associate with invariantsubunits of the CD3 signaling apparatus or complex. The intracellulartails of CD3 signaling subunits (e.g. CD3γ, CD3δ, CD3ε and CD3ζ chains)contain one or more immunoreceptor tyrosine-based activation motifs orITAMs that are involved in the signaling capacity of the TCR complex.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). A TCR may be cell-bound or in soluble form. In someembodiments, for purposes of the provided methods, the TCR is incell-bound form expressed on the surface of a cell. In some embodimentsa dTCR contains a first polypeptide wherein a sequence corresponding toa TCR a chain variable region sequence is fused to the N terminus of asequence corresponding to a TCR a chain constant region extracellularsequence, and a second polypeptide wherein a sequence corresponding to aTCR β chain variable region sequence is fused to the N terminus asequence corresponding to a TCR β chain constant region extracellularsequence, the first and second polypeptides being linked by a disulfidebond. In some embodiments, the bond can correspond to the nativeinterchain disulfide bond present in native dimeric αβ TCRs. In someembodiments, the interchain disulfide bonds are not present in a nativeTCR. For example, in some embodiments, one or more cysteines can beincorporated into the constant region extracellular sequences of dTCRpolypeptide pair. In some cases, both a native and a non-nativedisulfide bond may be desirable. In some embodiments, the TCR contains atransmembrane sequence to anchor to the membrane. In some embodiments, adTCR contains a TCR a chain containing a variable a domain, a constant adomain and a first dimerization motif attached to the C-terminus of theconstant a domain, and a TCR β chain comprising a variable β domain, aconstant β domain and a first dimerization motif attached to theC-terminus of the constant β domain, wherein the first and seconddimerization motifs easily interact to form a covalent bond between anamino acid in the first dimerization motif and an amino acid in thesecond dimerization motif linking the TCR a chain and TCR β chaintogether.

In some embodiments, the TCR is a scTCR, which is a single amino acidstrand containing an a chain and a β chain that is able to bind toMHC-peptide complexes. Typically, a scTCR can be generated using methodsknown to those of skill in the art, See e.g., International publishedPCT Nos. WO 96/13593, WO 96/18105, WO99/18129, WO04/033685,WO2006/037960, WO2011/044186; U.S. Pat. No. 7,569,664; and Schlueter, C.J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, a scTCRcontains a first segment constituted by an amino acid sequencecorresponding to a TCR a chain variable region, a second segmentconstituted by an amino acid sequence corresponding to a TCR β chainvariable region sequence fused to the N terminus of an amino acidsequence corresponding to a TCR β chain constant domain extracellularsequence, and a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment. In some embodiments, ascTCR contains a first segment constituted by an amino acid sequencecorresponding to a TCR β chain variable region, a second segmentconstituted by an amino acid sequence corresponding to a TCR a chainvariable region sequence fused to the N terminus of an amino acidsequence corresponding to a TCR a chain constant domain extracellularsequence, and a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment. In some embodiments, ascTCR contains a first segment constituted by an a chain variable regionsequence fused to the N terminus of an a chain extracellular constantdomain sequence, and a second segment constituted by a β chain variableregion sequence fused to the N terminus of a sequence β chainextracellular constant and transmembrane sequence, and, optionally, alinker sequence linking the C terminus of the first segment to the Nterminus of the second segment. In some embodiments, a scTCR contains afirst segment constituted by a TCR β chain variable region sequencefused to the N terminus of a β chain extracellular constant domainsequence, and a second segment constituted by an a chain variable regionsequence fused to the N terminus of a sequence a chain extracellularconstant and transmembrane sequence, and, optionally, a linker sequencelinking the C terminus of the first segment to the N terminus of thesecond segment. In some embodiments, for the scTCR to bind anMHC-peptide complex, the a and β chains must be paired so that thevariable region sequences thereof are orientated for such binding.Various methods of promoting pairing of an a and β in a scTCR are wellknown in the art. In some embodiments, a linker sequence is includedthat links the a and β chains to form the single polypeptide strand. Insome embodiments, the linker should have sufficient length to span thedistance between the C terminus of the a chain and the N terminus of theβ chain, or vice versa, while also ensuring that the linker length isnot so long so that it blocks or reduces bonding of the scTCR to thetarget peptide-MHC complex. In some embodiments, the linker of a scTCRsthat links the first and second TCR segments can be any linker capableof forming a single polypeptide strand, while retaining TCR bindingspecificity. In some embodiments, the linker sequence may, for example,have the formula -P-AA-P-, wherein P is proline and AA represents anamino acid sequence wherein the amino acids are glycine and serine. Insome embodiments, the first and second segments are paired so that thevariable region sequences thereof are orientated for such binding.Hence, in some cases, the linker has a sufficient length to span thedistance between the C terminus of the first segment and the N terminusof the second segment, or vice versa, but is not too long to block orreduces bonding of the scTCR to the target ligand. In some embodiments,the linker can contain from or from about 10 to 45 amino acids, such as10 to 30 amino acids or 26 to 41 amino acids residues, for example 29,30, 31 or 32 amino acids. In some embodiments, a scTCR contains adisulfide bond between residues of the single amino acid strand, which,in some cases, can promote stability of the pairing between the a and βregions of the single chain molecule (see e.g. U.S. Pat. No. 7,569,664).In some embodiments, the scTCR contains a covalent disulfide bondlinking a residue of the immunoglobulin region of the constant domain ofthe a chain to a residue of the immunoglobulin region of the constantdomain of the β chain of the single chain molecule. In some embodiments,the disulfide bond corresponds to the native disulfide bond present in anative dTCR. In some embodiments, the disulfide bond in a native TCR isnot present. In some embodiments, the disulfide bond is an introducednon-native disulfide bond, for example, by incorporating one or morecysteines into the constant region extracellular sequences of the firstand second chain regions of the scTCR polypeptide. Exemplary cysteinemutations include any as described above. In some cases, both a nativeand a non-native disulfide bond may be present.

In some embodiments, any of the TCRs, including a dTCR or scTCR, can belinked to signaling domains that yield an active TCR on the surface of aT cell. In some embodiments, the TCR is expressed on the surface ofcells. In some embodiments, the TCR does contain a sequencecorresponding to a transmembrane sequence. In some embodiments, thetransmembrane domain can be a Ca or CP transmembrane domain. In someembodiments, the transmembrane domain can be from a non-TCR origin, forexample, a transmembrane region from CD3z, CD28 or B7.1. In someembodiments, the TCR does contain a sequence corresponding tocytoplasmic sequences. In some embodiments, the TCR contains a CD3zsignaling domain. In some embodiments, the TCR is capable of forming aTCR complex with CD3. In some embodiments, the TCR or antigen bindingportion thereof may be a recombinantly produced natural protein ormutated form thereof in which one or more property, such as bindingcharacteristic, has been altered. In some embodiments, a TCR may bederived from one of various animal species, such as human, mouse, rat,or other mammal.

In some embodiments, the TCR comprises affinity to a target antigen on atarget cell. The target antigen may include any type of protein, orepitope thereof, associated with the target cell. For example, the TCRmay comprise affinity to a target antigen on a target cell thatindicates a particular disease state of the target cell. In someembodiments, the target antigen is processed and presented by MHCs.

In an exemplary embodiment, the target cell antigen is a New Yorkesophageal-1 (NY-ESO-1) peptide. NY-ESO-1 belongs to the cancer-testis(CT) antigen group of proteins. NY-ESO-1 is a highly immunogenic antigenin vitro and is presented to T cells via the MHC. CTLs recognizing theA2 presented epitope NY-ESO₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO:1), have beengrown from the blood and lymph nodes of myeloma patients. T cell clonesspecific for this epitope have been shown to kill tumor cells. A highaffinity TCR recognizing the NY-ESO₁₅₇₋₁₆₅ epitope may recognizeHLA-A2-positive, NY-ESO-1 positive cell lines (but not cells that lackeither HLA-A2 or NY-ESO). Accordingly, a TCR of the present disclosuremay be a HLA-A2-restricted NY-ESO-1 (SLLMWITQC; SEQ ID NO:1)-specificTCR. In one embodiment, an NY-ESO-1 TCR of the present disclosure is awild-type NY-ESO-1 TCR. A wild-type NY-ESO-1 TCR may include, withoutlimitation, the 8F NY-ESO-1 TCR (also referred to herein as “8F” or “8FTCR”), and the 1G4 NY-ESO-1 TCR (also referred to herein as “1G4” or“1G4 TCR”). In one embodiment, an NY-ESO-1 TCR of the present disclosureis an affinity enhanced 1G4 TCR, also called Ly95. 1G4 TCR and affinityenhanced 1G4 TCR is described in U.S. Pat. No. 8,143,376.

As described herein a TCR of the present disclosure may comprise a TCRalpha chain and a TCR beta chain. For example, a TCR having affinity forNY-ESO-1 comprises a TCR alpha chain and a TCR beta chain that together,contribute to the affinity of the TCR for NY-ESO-1. In one embodiment, aTCR having affinity for NY-ESO-1 comprises an 8F TCR alpha chaincomprising an 8F TCR alpha chain variable region comprising the aminoacid sequence set forth below:EEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGAGKSTFGDGTTLTVKPN (SEQ ID NO:2), which isencoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 3) GAGGAGGACCCCCAGGCCCTGTCCATCCAGGAGGGGGAGAATGCCACCATGAATTGCAGTTACAAGACTTCCATAAACAACCTGCAGTGGTACCGCCAGAACTCCGGCCGCGGCCTGGTGCACCTGATCCTCATCCGGTCGAATGAAAGGGAAAAGCACTCGGGACGCCTGCGAGTGACTCTGGACACGTCCAAGAAGTCGTCCAGTCTCTTAATCACCGCCTCTCGCGCAGCCGATACCGCATCGTACTTCTGTGCAACCGACGGGGGGGGCAAGAGTACATTCGGCGACGGCACTACCCTGACCGTGAAGCCAAAT.

Tolerable variations of the 8F TCR alpha chain of a TCR having affinityfor NY-ESO-1 will be known to those of skill in the art, whilemaintaining affinity to NY-ESO-1. For example, a TCR having affinity forNY-ESO-1 comprises an 8F TCR alpha chain variable region comprising anamino acid sequence that has at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the 8F TCR alpha chain variable regionamino acid sequence set forth in SEQ ID NO:2. In one embodiment, the TCRhaving affinity for NY-ESO-1 comprises the 8F TCR alpha chain variableregion comprising the amino acid sequence set forth in SEQ ID NO:2.

A TCR having affinity for NY-ESO-1 may comprise an 8F TCR alpha chainvariable region encoded by a nucleic acid sequence that has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least82%, at least 84%, at least 86%, at least 88%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to the8F TCR alpha chain variable region nucleic acid sequence set forth inSEQ ID NO:3. In one embodiment, the TCR having affinity for NY-ESO-1comprises the 8F TCR alpha chain variable region comprising the nucleicacid sequence set forth in SEQ ID NO:3.

The TCR alpha chain variable region of a TCR having affinity forNY-ESO-1 comprises three complementarity-determining regions (CDRs). Asused herein, a “complementarity-determining region” or “CDR” refers to aregion of the variable chain of an antigen binding molecule (such asimmunoglobulins and TCRs) that binds to a specific antigen. Accordingly,a TCR having affinity for NY-ESO-1 may comprise an 8F TCR alpha chainvariable region that comprises a CDR1 represented by the amino acidsequence TSINN (SEQ ID NO:4); a CDR2 represented by the amino acidsequence IRS; and a CDR3 represented by the amino acid sequence ATD.Tolerable variations to the CDRs of the TCR alpha chain variable regionof a TCR having affinity for NY-ESO-1 will be known to those of skill inthe art, while maintaining affinity to NY-ESO-1. For example, a TCRhaving affinity for NY-ESO-1 may comprise an 8F TCR alpha chain variableregion comprising a CDR1 that comprises an amino acid sequence that hasat least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the CDR1 amino acid sequence set forth in SEQ IDNO:4. For example, a TCR having affinity for NY-ESO-1 may comprise an 8FTCR alpha chain variable region comprising a CDR2 that comprises anamino acid sequence that has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity to the CDR2 represented by theamino acid sequence IRS. For example, a TCR having affinity for NY-ESO-1may comprise an 8F TCR alpha chain variable region comprising a CDR3that comprises an amino acid sequence that has at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to theCDR3 represented by the amino acid sequence ATD. In one embodiment, theTCR alpha chain variable region of a TCR having affinity for NY-ESO-1comprises the three aforementioned 8F TCR alpha chain variable regioncomplementarity-determining regions (CDRs).

An 8F TCR alpha chain of a TCR having affinity for NY-ESO-1 may comprisethe amino acid sequence set forth below:

(SEQ ID NO: 5) METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGAGKSTFGDGTTLTVKPNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS,which is encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 6) ATGGACTCGTGGACCTTATGCTGCGTGTCCCTGTGCATACTGGTTGCCAAGCACACAGACGCCGGGGTGATCCAGAGCCCCCGGCACGAAGTTACCGAGATGGGCCAGGAGGTGACGCTCCGATGCAAGCCCATCAGTGGCCACGATTATCTCTTCTGGTACCGCCAAACCATGATGCGCGGCTTGGAACTCCTCATCTACTTCAACAACAACGTCCCCATCGATGACTCCGGCATGCCTGAGGACAGGTTCAGTGCGAAGATGCCGAATGCATCCTTCTCCACCCTGAAGATACAGCCGAGTGAGCCCCGCGACTCCGCTGTGTACTTCTGCGCCTCTACTATCGGCGCCCAGCCTCAACATTTCGGCGACGGCACGCGCCTCAGTATCCTGGAGGACCTGAACAAGGTGTTCCCTCCGGAAGTGGCTGTGTTTGAGCCCTCCGAGGCAGAAATCTCACACACACAGAAGGCAACCCTCGTGTGTCTGGCAACAGGTTTCTTCCCAGATCACGTGGAGCTGAGTTGGTGGGTCAACGGCAAGGAGGTCCATAGCGGGGTGAGTACCGACCCACAGCCTCTCAAGGAGCAGCCTGCCCTCAACGACAGTAGGTACTGCCTGTCCTCGCGCCTCCGCGTGTCCGCAACGTTCTGGCAGAATCCCCGCAACCACTTCCGGTGCCAGGTCCAATTCTACGGCCTGAGTGAGAACGATGAGTGGACACAGGATAGGGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCCGCGCTGACTGCGGCTTCACCTCCGTGTCGTATCAGCAGGGCGTATTATCAGCCACCATTCTTTACGAAATCCTCCTCGGCAAGGCCACACTATACGCCGTGCTGGTGTCGGCGCTGGTGTTAATGGCGATGGTCAAGCGAAAGGATTAA.

An 8F TCR alpha chain having affinity for NY-ESO-1 may comprise an aminoacid sequence that has at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the 8F TCR alpha chain amino acidsequence set forth in SEQ ID NO:5. In one embodiment, the TCR havingaffinity for NY-ESO-1 comprises the 8F TCR alpha chain comprising theamino acid sequence set forth in SEQ ID NO:5. A TCR having affinity forNY-ESO-1 may comprise an 8F TCR alpha chain encoded by a nucleic acidsequence that has at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity to the 8F TCR alpha chain nucleic acid sequenceset forth in SEQ ID NO:6. In one embodiment, the TCR having affinity forNY-ESO-1 comprises the 8F TCR alpha chain comprising the nucleic acidsequence set forth in SEQ ID NO:6.

As described herein, a TCR of the present disclosure may comprise a TCRalpha chain and a TCR beta chain. In one embodiment, a TCR havingaffinity for NY-ESO-1 comprises an 8F TCR beta chain comprising an 8FTCR beta chain variable region comprising the amino acid sequence setforth below:

(SEQ ID NO: 7) VIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASTIGAQPQ HFGDGTRLSILE,which is encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 8) GTGATCCAGAGCCCCCGGCACGAAGTTACCGAGATGGGCCAGGAGGTGACGCTCCGATGCAAGCCCATCAGTGGCCACGATTATCTCTTCTGGTACCGCCAAACCATGATGCGCGGCTTGGAACTCCTCATCTACTTCAACAACAACGTCCCCATCGATGACTCCGGCATGCCTGAGGACAGGTTCAGTGCGAAGATGCCGAATGCATCCTTCTCCACCCTGAAGATACAGCCGAGTGAGCCCCGCGACTCCGCTGTGTACTTCTGCGCCTCTACTATCGGCGCCCAGCCTCAACATTTCGGCGACGGCACGCGCCTCAGTATCCTGGAG.

Tolerable variations of the TCR alpha chain of a TCR having affinity forNY-ESO-1 will be known to those of skill in the art, while maintainingaffinity to NY-ESO-1. For example, a TCR having affinity for NY-ESO-1comprises an 8F TCR beta chain variable region comprising an amino acidsequence that has at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity to the 8F TCR beta chain variable region aminoacid sequence set forth in SEQ ID NO:7. In one embodiment, the TCRhaving affinity for NY-ESO-1 comprises the 8F TCR beta chain variableregion comprising the amino acid sequence set forth in SEQ ID NO:7.

A TCR having affinity for NY-ESO-1 may comprise an 8F TCR beta chainvariable region encoded by a nucleic acid sequence that has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least82%, at least 84%, at least 86%, at least 88%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to the8F TCR beta chain variable region nucleic acid sequence set forth in SEQID NO:8. In one embodiment, the TCR having affinity for NY-ESO-1comprises the 8F TCR beta chain variable region comprising the nucleicacid sequence set forth in SEQ ID NO:8.

The TCR beta chain variable region of a TCR having affinity for NY-ESO-1comprises three complementarity-determining regions (CDRs). Accordingly,a TCR having affinity for NY-ESO-1 may comprise an 8F TCR beta chainvariable region that comprises a CDR1 represented by the amino acidsequence SGHDY (SEQ ID NO:9); a CDR2 represented by the amino acidsequence FNNNVP (SEQ ID NO:10); and a CDR3 represented by the amino acidsequence ASTI (SEQ ID NO:11). Tolerable variations to the CDRs of theTCR beta chain variable region of a TCR having affinity for NY-ESO-1will be known to those of skill in the art, while maintaining affinityto NY-ESO-1. For example, a TCR having affinity for NY-ESO-1 maycomprise an 8F TCR beta chain variable region comprising a CDR1 thatcomprises an amino acid sequence that has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% sequence identity to the CDR1amino acid sequence set forth in SEQ ID NO:9. For example, a TCR havingaffinity for NY-ESO-1 may comprise an 8F TCR beta chain variable regioncomprising a CDR2 that comprises an amino acid sequence that has atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the CDR2 amino acid sequence set forth in SEQ IDNO:10. For example, a TCR having affinity for NY-ESO-1 may comprise an8F TCR beta chain variable region comprising a CDR3 that comprises anamino acid sequence that has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity to the CDR3 amino acidsequence set forth in SEQ ID NO:11. In one embodiment, the TCR betachain variable region of an 8F TCR having affinity for NY-ESO-1comprises the three aforementioned TCR beta chain variable regioncomplementarity-determining regions (CDRs).

An 8F TCR beta chain of a TCR having affinity for NY-ESO-1 may comprisethe amino acid sequence set forth below:

(SEQ ID NO: 12) METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGAGKSTFGDGTTLTVKPNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS,which is encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 13) ATGGAGACCCTGCTCGGGGTCTCACTGGTCATCCTGTGGCTGCAGCTGGCCAGGGTGAACTCGCAGCAGGGGGAGGAGGACCCCCAGGCCCTGTCCATCCAGGAGGGGGAGAATGCCACCATGAATTGCAGTTACAAGACTTCCATAAACAACCTGCAGTGGTACCGCCAGAACTCCGGCCGCGGCCTGGTGCACCTGATCCTCATCCGGTCGAATGAAAGGGAAAAGCACTCGGGACGCCTGCGAGTGACTCTGGACACGTCCAAGAAGTCGTCCAGTCTCTTAATCACCGCCTCTCGCGCAGCCGATACCGCATCGTACTTCTGTGCAACCGACGGGGGGGGCAAGAGTACATTCGGCGACGGCACTACCCTGACCGTGAAGCCAAATATCCAGAAGCCTGATCCAGCTGTCTATCAGTTGCGCGATTCCAAATCGTCTGACAAATCTGTGTGCCTGTTCACCGACTTCGACTCCCAGACGAACGTGTCCCAGAGTAAAGACAGCGACGTGTACATCACTGATAAGACCGTGCTGGACATGCGCTCCATGGACTTTAAAAGTAACAGCGCTGTAGCGTGGAGCAACAAGAGTGACTTCGCCTGCGCCAACGCCTTCAATAACTCTATCATACCTGCCGATACCTTCTTCCCGAGCCCCGAATCCAGTTGCGACGTGAAGCTCGTGGAGAAGAGCTTTGAGACAGACACCAACCTGAACTTCCAAAACCTGTCCGTCATTGGCTTCAGGATCCTCCTCCTCAAGGTGGCCGGCTTCAACTTGCTCATGACGCTGAGACTCTGGAGTTCA.

An 8F TCR beta chain having affinity for NY-ESO-1 may comprise an aminoacid sequence that has at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the 8F TCR beta chain amino acid sequenceset forth in SEQ ID NO:12. In one embodiment, the TCR having affinityfor NY-ESO-1 comprises the 8F TCR beta chain comprising the amino acidsequence set forth in SEQ ID NO:12. A TCR having affinity for NY-ESO-1may comprise an 8F TCR beta chain encoded by a nucleic acid sequencethat has at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the 8F TCR beta chain nucleic acid sequence setforth in SEQ ID NO:13. In one embodiment, the TCR having affinity forNY-ESO-1 comprises the 8F TCR beta chain comprising the nucleic acidsequence set forth in SEQ ID NO:13.

C. Switch Receptors

The present invention provides a switch receptor for use in the modifiedcell of the invention. As used herein, the term “switch receptor” or“chimeric switch receptor” refers to a molecule designed to switch anegative signal transduction signal into a positive signal. In someembodiments, the switch receptor is a chimeric protein comprising afirst protein or fragment thereof associated with a negative signal, anda second protein or fragment thereof associated with a positive signal.Examples of proteins associated with a negative signal include, withoutlimitation, CTLA-4, PD-1, BTLA, TIM-3 and the like. Examples of proteinsassociated with a positive signal include, without limitation, CD28,ICOS, 4-1 BB, TGFβR and the like.

Accordingly, a switch receptor, when expressed in a cell (e.g.,mammalian cell), converts a negative signal into a positive signal inthe cell. In some embodiments, a switch receptor of the presentdisclosure comprises a first domain derived from a protein or fragmentthereof that delivers a negative signal; and a second domain derivedfrom a protein or fragment thereof that delivers a positive signal.

Suitable first domains derived from a protein or fragment thereof thatdelivers a negative signal include, variants or derivatives of wild-typeCTLA-4. In one embodiment, the first domain of the switch receptorcomprises at least a portion of the extracellular domain of the CTLA-4protein, specifically that portion of the extracellular domain which isnecessary for binding to the natural ligand of CTLA-4. Variants of thewild-type form of the extracellular domain, or the portion of theextracellular domain responsible for binding to the natural ligand ofCTLA-4, are also included in the present invention. Suitable firstdomains derived from a protein or fragment thereof that delivers anegative signal include, variants or derivatives of wild-type PD-1. Inone embodiment, the first domain of the switch receptor comprises atleast a portion of the extracellular domain of the PD-1 protein,specifically that portion of the extracellular domain which is necessaryfor binding to the natural ligand of PD-1. Variants of the wild-typeform of the extracellular domain, or the portion of the extracellulardomain responsible for binding to the natural ligand of PD-1, are alsoincluded in the present invention. For example, a variant PD1extracellular domain having an A132L substitution relative to the fulllength amino acid sequence of PD1, is included in the present invention.Suitable first domains derived from a protein or fragment thereof thatdelivers a negative signal include, variants or derivatives of wild-typeBTLA. In one embodiment, the first domain of the switch receptorcomprises at least a portion of the extracellular domain of the BTLAprotein, specifically that portion of the extracellular domain which isnecessary for binding to the natural ligand of BTLA. Variants of thewild-type form of the extracellular domain, or the portion of theextracellular domain responsible for binding to the natural ligand ofBTLA, are also included in the present invention. Suitable first domainsderived from a protein or fragment thereof that delivers a negativesignal include, variants or derivatives of wild-type TGFβR (e.g., TGFβRIor TGFβRII). In one embodiment, the first domain of the switch receptorcomprises at least a portion of the extracellular domain of the TGFβRprotein (e.g., TGFβRI or TGFβRII), specifically that portion of theextracellular domain which is necessary for binding to the naturalligand of TGFβR (e.g., TGFβRI or TGFβRII). Variants of the wild-typeform of the extracellular domain, or the portion of the extracellulardomain responsible for binding to the natural ligand of TGFβR (e.g.,TGFβRI or TGFβRII), are also included in the present invention. Suitablefirst domains derived from a protein or fragment thereof that delivers anegative signal include, variants or derivatives of wild-type TIM3. Inone embodiment, the first domain of the switch receptor comprises atleast a portion of the extracellular domain of the TIM3 protein,specifically that portion of the extracellular domain which is necessaryfor binding to the natural ligand of TIM3. Variants of the wild-typeform of the extracellular domain, or the portion of the extracellulardomain responsible for binding to the natural ligand of TIM3, are alsoincluded in the present invention.

Suitable second domains derived from a protein or fragment thereof thatdelivers a negative signal include, variants or derivatives of the ICOSprotein. In one embodiment, the second domain of the switch receptorcomprises at least a portion of the intracellular domain (also referredto as endodomain or cytoplasmic domain) of the ICOS protein,specifically that portion which is necessary for triggering a signal tointracellular components of the cell. Variants of the wild-type form ofthe intracellular domain of the ICOS protein, or the portion of theintracellular domain responsible for signaling, are also included in thepresent invention. Suitable second domains derived from a protein orfragment thereof that delivers a negative signal include, variants orderivatives of the CD28 protein. In one embodiment, the second domain ofthe switch receptor comprises at least a portion of the intracellulardomain (also referred to as endodomain or cytoplasmic domain) of theCD28 protein, specifically that portion which is necessary fortriggering a signal to intracellular components of the cell. Variants ofthe wild-type form of the intracellular domain of the CD28 protein, orthe portion of the intracellular domain responsible for signaling, arealso included in the present invention. Suitable second domains derivedfrom a protein or fragment thereof that delivers a negative signalinclude, variants or derivatives of IL-12R (e.g., IL-12Rβ1 or IL-12Rβ2).In one embodiment, the second domain of the switch receptor comprises atleast a portion of the intracellular domain (also referred to asendodomain or cytoplasmic domain) of IL-12R (e.g., IL-12Rβ1 orIL-12Rβ2), specifically that portion which is necessary for triggering asignal to intracellular components of the cell. Variants of thewild-type form of the intracellular domain of IL-12R (e.g., IL-12Rβ1 orIL-12Rβ2), or the portion of the intracellular domain responsible forsignaling, are also included in the present invention. Suitable seconddomains derived from a protein or fragment thereof that delivers anegative signal include, variants or derivatives of the 4-1BB protein.In one embodiment, the second domain of the switch receptor comprises atleast a portion of the intracellular domain (also referred to asendodomain or cytoplasmic domain) of the 4-1BB protein, specificallythat portion which is necessary for triggering a signal to intracellularcomponents of the cell. Variants of the wild-type form of theintracellular domain of the 4-1 BB protein, or the portion of theintracellular domain responsible for signaling, are also included in thepresent invention.

A switch receptor suitable for use in the present invention comprises apolypeptide corresponding to a cytoplasmic, transmembrane andextracellular domain, as well as polypeptides corresponding to smallerportions of the cytoplasmic, transmembrane and extracellular domain. Inone embodiment the switch receptor comprises the transmembrane domain ofthe protein or fragment thereof that delivers a negative signal. Inanother embodiment, the switch receptor comprises the transmembranedomain of the protein or fragment thereof that delivers a positivesignal.

In one embodiment, a switch receptor suitable for use in the presentinvention is a PD1-CD28 switch receptor. The PD1-CD28 switch receptorcomprises a variant of the PD1 extracellular domain, a CD28transmembrane domain, and a CD28 cytoplasmic domain. In one embodiment,the PD1-CD28 switch receptor comprises an amino acid sequence set forthbelow:

(SEQ ID NO: 14) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS,encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 15) ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC.

Tolerable variations of the PD1-CD28 switch receptor will be known tothose of skill in the art, while maintaining its intended biologicalactivity (e.g., converting a negative PD1 signal into a positive CD28signal when expressed in a cell). Accordingly, a PD1-CD28 switchreceptor of the present invention may comprise an amino acid sequencethat has at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the PD1-CD28 switch receptor amino acid sequenceset forth in SEQ ID NO:14. Accordingly, a PD1-CD28 switch receptor ofthe present invention may be encoded by a nucleic acid comprising anucleic acid sequence that has at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the PD1-CD28 switch receptor nucleic acidsequence set forth in SEQ ID NO:15.

In one embodiment, a switch receptor suitable for use in the presentinvention is a TIM3-CD28 switch receptor. The TIM3-CD28 switch receptorcomprises a TIM3 extracellular domain, a CD28 transmembrane domain, anda CD28 cytoplasmic domain. In one embodiment, the TIM3-CD28 switchreceptor comprises an amino acid sequence set forth below:

(SEQ ID NO: 132) MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS,encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 133) ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG ACTTCGCAGCCTATCGCTCC.

Tolerable variations of the TIM3-CD28 switch receptor will be known tothose of skill in the art, while maintaining its intended biologicalactivity (e.g., converting a negative TIM3 signal into a positive CD28signal when expressed in a cell). Accordingly, a TIM3-CD28 switchreceptor of the present invention may comprise an amino acid sequencethat has at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the TIM3-CD28 switch receptor amino acid sequenceset forth in SEQ ID NO:132. Accordingly, a TIM3-CD28 switch receptor ofthe present invention may be encoded by a nucleic acid comprising anucleic acid sequence that has at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the TIM3-CD28 switch receptor nucleicacid sequence set forth in SEQ ID NO:133.

In one embodiment, a switch receptor suitable for use in the presentinvention is a PD1-41BB switch receptor (also referred to herein asPD1.BB). The PD1-41 BB switch receptor comprises a PD1 extracellulardomain, a CD8alpha transmembrane domain, and a 4-1 BB cytoplasmicdomain. In one embodiment, the PD1-41BB switch receptor comprises anamino acid sequence set forth below:

(SEQ ID NO: 134) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 135) ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTG. 

Tolerable variations of the PD1-41BB switch receptor will be known tothose of skill in the art, while maintaining its intended biologicalactivity (e.g., converting a negative PD1 signal into a positive 4-1EEsignal when expressed in a cell). Accordingly, a PD1-41 BB switchreceptor of the present invention may comprise an amino acid sequencethat has at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the PD1-41BB switch receptor amino acid sequenceset forth in SEQ ID NO:134. Accordingly, a PD1-41BB switch receptor ofthe present invention may be encoded by a nucleic acid comprising anucleic acid sequence that has at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the PD1-41BB switch receptor nucleic acidsequence set forth in SEQ ID NO:135.

In one embodiment, a switch receptor suitable for use in the presentinvention is a PD1^(A132L)-41BB switch receptor (also referred to hereinas PD1^(A132L)PTM.BB or PD1*.BB). The PD1^(A132L)-41 BB switch receptorcomprises a variant PD1 extracellular domain having a A132L substitutionrelative to the full length amino acid sequence of PD1, a CD8alphatransmembrane domain, and a 4-1BB cytoplasmic domain. The PD1 A132Lsubstitution increases its affinity with PD-L1. See, e.g., Zhang et al.Immunity 2004, 20:337-347. In one embodiment, the PD1^(A132L)-41 BBswitch receptor comprises an amino acid sequence set forth below:

(SEQ ID NO: 136) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, encoded b the nucleic acid sequence set forth below:

(SEQ ID NO: 137) ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG  GATGTGAACTG.

Tolerable variations of the PD1^(A132L)-41 BB switch receptor will beknown to those of skill in the art, while maintaining its intendedbiological activity (e.g., converting a negative PD1 signal into apositive 4-1BB signal when expressed in a cell). Accordingly, aPD1^(A132L)-41BB switch receptor of the present invention may comprisean amino acid sequence that has at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% sequence identity to the PD1^(A132L)-41 BB switchreceptor amino acid sequence set forth in SEQ ID NO:136. Accordingly, aPD1^(A132L)-41BB switch receptor of the present invention may be encodedby a nucleic acid comprising a nucleic acid sequence that has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to thePD1^(A132L)-41BB switch receptor nucleic acid sequence set forth in SEQID NO:137.

In one embodiment, a switch receptor suitable for use in the presentinvention is a PD1^(A132L)-CD28 switch receptor (also referred to hereinas PD1^(A132L)PTM.CD28 or PD1*.CD28). The PD1^(A132L)-CD28 switchreceptor comprises a variant PD1 extracellular domain having a A132Lsubstitution relative to the full length amino acid sequence of PD1, aCD28 transmembrane domain, and a CD28 cytoplasmic domain. The PD1 A132Lsubstitution increases its affinity with PD-L1. See, e.g., Zhang et al.Immunity 2004, 20:337-347. In one embodiment, the PD1^(A132L)-CD28switch receptor comprises an amino acid sequence set forth below:

(SEQ ID NO: 138) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS, encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 139) ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC.

Tolerable variations of the PD1^(A132L)-CD28 switch receptor will beknown to those of skill in the art, while maintaining its intendedbiological activity (e.g., converting a negative PD1 signal into apositive CD28 signal when expressed in a cell). Accordingly, aPD1^(A132L)-CD28 switch receptor of the present invention may comprisean amino acid sequence that has at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% sequence identity to the PD1^(A132L)-CD28 switchreceptor amino acid sequence set forth in SEQ ID NO:138. Accordingly, aPD1^(A132L)-CD28 switch receptor of the present invention may be encodedby a nucleic acid comprising a nucleic acid sequence that has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to thePD1^(A132L)-CD28 switch receptor nucleic acid sequence set forth in SEQID NO:139.

In one embodiment, a switch receptor suitable for use in the presentinvention is a TGFβRI-IL-12Rβ1 switch receptor. In one embodiment, theTGFβRI-IL-12Rβ1 switch receptor comprises an amino acid sequence setforth below:

(SEQ ID NO: 16) LEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYIRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM, encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 17) CTGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCGGCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCAGGGCCGCACGGCACCTGTGCCCGCCGCTGCCCACACCCTGTGCCAGCTCCGCCATTGAGTTCCCTGGAGGGAAGGAGACTTGGCAGTGGATCAACCCAGTGGACTTCCAGGAAGAGGCATCCCTGCAGGAGGCCCTGGTGGTAGAGATGTCCTGGGACAAAGGCGAGAGGACTGAGCCTCTCGAGAAGACAGAGCTACCTGAGGGTGCCCCTGAGCTGGCCCTGGATACAGAGTTGTCCTTGGAGGATGGAGACAGGT GCAAGGCCAAGATGTGA.

Tolerable variations of the TGFβRI-IL-12Rβ1 switch receptor will beknown to those of skill in the art, while maintaining its intendedbiological activity (e.g., converting a negative TGFβ signal into apositive IL-12 signal when expressed in a cell). Accordingly, aTGFβRI-IL-12Rβ1 switch receptor of the present invention may comprise anamino acid sequence that has at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the TGFβRI-IL-12Rβ1 switch receptor aminoacid sequence set forth in SEQ ID NO:16. Accordingly, a TGFβRI-IL-12Rβ1switch receptor of the present invention may be encoded by a nucleicacid comprising a nucleic acid sequence that has at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% sequence identity to the TGFβRI-IL-12Rβ1switch receptor nucleic acid sequence set forth in SEQ ID NO:17.

In one embodiment, a switch receptor suitable for use in the presentinvention is a TGFβRII-IL-12Rβ2 switch receptor. In one embodiment, theTGFβRII-IL-12Rβ2 switch receptor comprises an amino acid sequence setforth below:

(SEQ ID NO: 18) MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDS LML, encoded b the nucleic acid sequence set forth below:

(SEQ ID NO: 19) ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACCAGCAAAAGGTGTTTGTTCTCCTAGCAGCCCTCAGACCTCAGTGGTGTAGCAGAGAAATTCCAGATCCAGCAAATAGCACTTGCGCTAAGAAATATCCCATTGCAGAGGAGAAGACACAGCTGCCCTTGGACAGGCTCCTGATAGACTGGCCCACGCCTGAAGATCCTGAACCGCTGGTCATCAGTGAAGTCCTTCATCAAGTGACCCCAGTTTTCAGACATCCCCCCTGCTCCAACTGGCCACAAAGGGAAAAAGGAATCCAAGGTCATCAGGCCTCTGAGAAAGACATGATGCACAGTGCCTCAAGCCCACCACCTCCAAGAGCTCTCCAAGCTGAGAGCAGACAACTGGTGGATCTGTACAAGGTGCTGGAGAGCAGGGGCTCCGACCCAAAGCCAGAAAACCCAGCCTGTCCCTGGACGGTGCTCCCAGCAGGTGACCTTCCCACCCATGATGGCTACTTACCCTCCAACATAGATGACCTCCCCTCACATGAGGCACCTCTCGCTGACTCTCTGGAAGAACTGGAGCCTCAGCACATCTCCCTTTCTGTTTTCCCCTCAAGTTCTCTTCACCCACTCACCTTCTCCTGTGGTGATAAGCTGACTCTGGATCAGTTAAAGATGAGGTGTGACTCC CTCATGCTCTGA.

Tolerable variations of the TGFβRII-IL-12Rβ2 switch receptor will beknown to those of skill in the art, while maintaining its intendedbiological activity (e.g., converting a negative TGFβ signal into apositive IL-12 signal when expressed in a cell). Accordingly, aTGFβRII-IL-12Rβ2 switch receptor of the present invention may comprisean amino acid sequence that has at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% sequence identity to the TGFβRII-IL-12Rβ2 switchreceptor amino acid sequence set forth in SEQ ID NO:18. Accordingly, aTGFβRII-IL-12Rβ2 switch receptor of the present invention may be encodedby a nucleic acid comprising a nucleic acid sequence that has at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity to theTGFβRII-IL-12Rβ2 switch receptor nucleic acid sequence set forth in SEQID NO:19.

Other TGFβ signal converters or inhibitors are suitable for use in thepresent invention. In one embodiment, a dominant negative TGFβRII“switch receptor” (TGFβRIIDN) is suitable for use in the presentinvention. In such embodiment, the switch receptor is a truncatedvariant of a protein associated with a negative signal (e.g., TGFβRII).In one embodiment, the TGFβRIIDN comprises an amino acid sequence setforth below:

(SEQ ID NO: 20) MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSS G, encoded by the nucleic acid sequence set forth below:

(SEQ ID NO: 21) ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCC  GGA.

Tolerable variations of the TGFβRIIDN will be known to those of skill inthe art, while maintaining its intended biological activity.Accordingly, a TGFβRIIDN of the present invention may comprise an aminoacid sequence that has at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the TGFβRIIDN amino acid sequence setforth in SEQ ID NO:20. Accordingly, a TGFβRIIDN of the present inventionmay be encoded by a nucleic acid comprising a nucleic acid sequence thathas at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% sequenceidentity to the TGFβRIIDN nucleic acid sequence set forth in SEQ IDNO:21. Other suitable switch receptors for use in the present inventionare described in PCT Publication No. WO2013019615A2, the disclosure ofwhich is incorporated herein by reference.

D. Nucleic Acids and Expression Vectors

The present disclosure provides a nucleic acid encoding an exogenous TCRand/or a switch receptor. In one embodiment, a nucleic acid of thepresent disclosure comprises a nucleic acid sequence encoding anexogenous TCR (e.g., an NY-ESO-1 TCR). In one embodiment, a nucleic acidof the present disclosure comprises a nucleic acid sequence encoding aswitch receptor (e.g., a PD1-CD28 switch receptor).

In some embodiments, a nucleic acid of the present disclosure providesfor the production of a TCR as described herein, e.g., in a mammaliancell. In some embodiments, a nucleic acid of the present disclosureprovides for amplification of the TCR-encoding nucleic acid.

As described herein, a TCR of the present disclosure comprises a TCRalpha chain and a TCR beta chain. Accordingly, the present disclosureprovides a nucleic acid encoding a TCR alpha chain, and a nucleic acidencoding a TCR beta chain. In some embodiments, the nucleic acidencoding a TCR alpha chain is separate from the nucleic acid encoding aTCR beta chain. In an exemplary embodiment, the nucleic acid encoding aTCR alpha chain, and the nucleic acid encoding a TCR beta chain, resideswithin the same nucleic acid.

In some embodiments, a nucleic acid of the present disclosure comprisesa nucleic acid comprising a TCR alpha chain coding sequence and a TCRbeta chain coding sequence. In some embodiments, a nucleic acid of thepresent disclosure comprises a nucleic acid comprising a TCR alpha chaincoding sequence and a TCR beta chain coding sequence that is separatedby a linker. A linker for use in the present disclosure allows formultiple proteins to be encoded by the same nucleic acid sequence (e.g.,a multicistronic or bicistronic sequence), which are translated as apolyprotein that is dissociated into separate protein components. Forexample, a linker for use in a nucleic acid of the present disclosurecomprising a TCR alpha chain coding sequence and a TCR beta chain codingsequence, allows for the TCR alpha chain and TCR beta chain to betranslated as a polyprotein that is dissociated into separate TCR alphachain and TCR beta chain components.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for an internal ribosome entry site (IRES). As used herein, “aninternal ribosome entry site” or “IRES” refers to an element thatpromotes direct internal ribosome entry to the initiation codon, such asATG, of a protein coding region, thereby leading to cap-independenttranslation of the gene. Various internal ribosome entry sites are knownto those of skill in the art, including, without limitation, IRESobtainable from viral or cellular mRNA sources, e.g., immunoglobulinheavy-chain binding protein (BiP); vascular endothelial growth factor(VEGF); fibroblast growth factor 2; insulin-like growth factor;translational initiation factor eIF4G; yeast transcription factors TFIIDand HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus,aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloneymurine leukemia virus (MoMLV). Those of skill in the art would be ableto select the appropriate IRES for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for a self-cleaving peptide. As used herein, a “self-cleavingpeptide” or “2A peptide” refers to an oligopeptide that allows multipleproteins to be encoded as polyproteins, which dissociate into componentproteins upon translation. Use of the term “self-cleaving” is notintended to imply a proteolytic cleavage reaction. Various self-cleavingor 2A peptides are known to those of skill in the art, including,without limitation, those found in members of the Picornaviridae virusfamily, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis Avirus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1(PTV-1); and carioviruses such as Theilovirus and encephalomyocarditisviruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV arereferred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.Those of skill in the art would be able to select the appropriateself-cleaving peptide for use in the present invention.

In some embodiments, a linker further comprises a nucleic acid sequencethat encodes a furin cleavage site. Furin is a ubiquitously expressedprotease that resides in the trans-golgi and processes proteinprecursors before their secretion. Furin cleaves at the COOH— terminusof its consensus recognition sequence. Various furin consensusrecognition sequences (or “furin cleavage sites”) are known to those ofskill in the art, including, without limitation, Arg-X-Lys-Arg (SEQ IDNO:22) or Arg-X-Arg-Arg (SEQ ID NO:23), (Lys/Arg)-Arg-X-(Lys/Arg)-Arg(SEQ ID NO:24) and Arg-X-X-Arg (SEQ ID NO:25), such as anArg-Gln-Lys-Arg (SEQ ID NO:26), where X is any naturally occurring aminoacid. Those of skill in the art would be able to select the appropriateFurin cleavage site for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequenceencoding a combination of a Furin cleavage site and a 2A peptide.Examples include, without limitation, a linker comprising a nucleic acidsequence encoding Furin and F2A, a linker comprising a nucleic acidsequence encoding Furin and E2A, a linker comprising a nucleic acidsequence encoding Furin and P2A, a linker comprising a nucleic acidsequence encoding Furin and T2A. Those of skill in the art would be ableto select the appropriate combination for use in the present invention.In such embodiments, the linker may further comprise a spacer sequencebetween the Furin and 2A peptide. Various spacer sequences are known inthe art, including, without limitation, glycine serine (GS) spacers suchas (GS)n, (GSGGS)n (SEQ ID NO:27) and (GGGS)n (SEQ ID NO:28), where nrepresents an integer of at least 1. Exemplary spacer sequences cancomprise amino acid sequences including, without limitation, GGSG (SEQID NO:29), GGSGG (SEQ ID NO:30), GSGSG (SEQ ID NO:31), GSGGG (SEQ IDNO:32), GGGSG (SEQ ID NO:33), GSSSG (SEQ ID NO:34), and the like. Thoseof skill in the art would be able to select the appropriate spacersequence for use in the present invention.

In an exemplary embodiment, a nucleic acid of the present disclosurecomprises a nucleic acid sequence comprising a TCR alpha chain codingsequence and a TCR beta chain coding sequence that is separated by aFurin-(G4S)2-T2A (F-GS2-T2A) linker. The F-GS2-T2A linker may be encodedby the nucleic acid sequenceCGTGCGAAGAGGGGCGGCGGGGGCTCCGGCGGGGGAGGCAGTGAGGGCCGCGGCTCCCTGCTGACCTGCGGAGATGTAGAAGAGAACCCAGGCCCC (SEQ ID NO:35), and may comprisethe amino acid sequence RAKRGGGGSGGGGSEGRGSLLTCGDVEENPGP (SEQ ID NO:36).Those of skill in the art would appreciate that linkers of the presentinvention may include tolerable sequence variations.

In some embodiments, the present disclosure provides a nucleic acidcomprising a nucleic acid sequence encoding a switch receptor asdescribed herein. In some embodiments, a nucleic acid comprises anucleic acid sequence encoding a switch receptor and a nucleic acidsequence encoding a TCR (e.g., NY-ESO-1 TCR). In one embodiment, thenucleic acid sequence encoding the switch receptor and the nucleic acidsequence encoding the TCR resides on separate nucleic acids. In oneembodiment, the nucleic acid sequence encoding the switch receptor andthe nucleic acid sequence encoding the TCR resides within the samenucleic acid. In such an embodiment, the nucleic acid sequence encodingthe switch receptor and the nucleic acid sequence encoding the TCR isseparated by a linker as described herein.

For example, a nucleic acid of the present disclosure may comprise anucleic acid sequence encoding a switch receptor, a linker, and anucleic acid sequence encoding a TCR. In one embodiment, the linkercomprises a nucleic acid sequence encoding a 2A peptide (e.g., F2A). Inan exemplary embodiment, a nucleic acid of the present disclosure maycomprise a nucleic acid sequence encoding a switch receptor and anucleic acid sequence encoding a TCR separated by a nucleic acidsequence encoding F2A. In an exemplary embodiment, the nucleic acidsequence encoding a TCR comprises a TCR alpha chain coding sequence anda TCR beta chain coding sequence separated by a nucleic acid sequenceencoding F-GS2-T2A.

Accordingly, in one embodiment, a nucleic acid of the present disclosurecomprises from 5′ to 3′: a nucleic acid sequence encoding a switchreceptor, a nucleic acid sequence encoding a linker, and a nucleic acidsequence encoding a TCR. In one embodiment, a nucleic acid of thepresent disclosure comprises from 5′ to 3′: a nucleic acid sequenceencoding a TCR, a nucleic acid sequence encoding a linker, and a nucleicacid sequence encoding a switch receptor. In an exemplary embodiment, anucleic acid of the present disclosure comprises from 5′ to 3′: anucleic acid sequence encoding a switch receptor, a nucleic acidsequence encoding F2A, and a nucleic acid sequence encoding a TCR. Inanother exemplary embodiment, a nucleic acid of the present disclosurecomprises from 5′ to 3′: a nucleic acid sequence encoding a switchreceptor, a nucleic acid sequence encoding F2A, a nucleic acid sequenceencoding a TCR alpha chain, a nucleic acid sequence encoding F-GS2-T2A,and a nucleic acid sequence encoding a TCR beta chain.

In some embodiments, a nucleic acid of the present disclosure may beoperably linked to a transcriptional control element, e.g., a promoter,and enhancer, etc. Suitable promoter and enhancer elements are known tothose of skill in the art.

For expression in a bacterial cell, suitable promoters include, but arenot limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. Forexpression in a eukaryotic cell, suitable promoters include, but are notlimited to, light and/or heavy chain immunoglobulin gene promoter andenhancer elements; cytomegalovirus immediate early promoter; herpessimplex virus thymidine kinase promoter; early and late SV40 promoters;promoter present in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. Suitable reversible promoters, including reversible induciblepromoters are known in the art. Such reversible promoters may beisolated and derived from many organisms, e.g., eukaryotes andprokaryotes. Modification of reversible promoters derived from a firstorganism for use in a second organism, e.g., a first prokaryote and asecond a eukaryote, a first eukaryote and a second a prokaryote, etc.,is well known in the art. Such reversible promoters, and systems basedon such reversible promoters but also comprising additional controlproteins, include, but are not limited to, alcohol regulated promoters(e.g., alcohol dehydrogenase I (alcA) gene promoter, promotersresponsive to alcohol transactivator proteins (A1cR), etc.),tetracycline regulated promoters, (e.g., promoter systems includingTetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g.,rat glucocorticoid receptor promoter systems, human estrogen receptorpromoter systems, retinoid promoter systems, thyroid promoter systems,ecdysone promoter systems, mifepristone promoter systems, etc.), metalregulated promoters (e.g., metallothionein promoter systems, etc.),pathogenesis-related regulated promoters (e.g., salicylic acid regulatedpromoters, ethylene regulated promoters, benzothiadiazole regulatedpromoters, etc.), temperature regulated promoters (e.g., heat shockinducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter,etc.), light regulated promoters, synthetic inducible promoters, and thelike.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an NcrI (p46) promoter; see, e.g., Eckelhart et al. Blood (2011)117:1565.

For expression in a yeast cell, a suitable promoter is a constitutivepromoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, aPYK1 promoter and the like; or a regulatable promoter such as a GAL1promoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDHpromoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use inPichia). Selection of the appropriate vector and promoter is well withinthe level of ordinary skill in the art. Suitable promoters for use inprokaryotic host cells include, but are not limited to, a bacteriophageT7 RNA polymerase promoter; a trp promoter; a lac operon promoter; ahybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybridpromoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tacpromoter, and the like; an araBAD promoter; in vivo regulated promoters,such as an ssaG promoter or a related promoter (see, e.g., U.S. PatentPublication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J.Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl.Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne etal. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan etal., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004)22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888-892); asigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBankAccession Nos. AX798980, AX798961, and AX798183); a stationary phasepromoter, e.g., a dps promoter, an spv promoter, and the like; apromoter derived from the pathogenicity island SPI-2 (see, e.g.,WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect.Immun. (2002) 70:1087-1096); an rpsM promoter (see, e.g., Valdivia andFalkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g.,Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U.(eds), Topics in Molecular and Structural Biology, Protein—Nucleic AcidInteraction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035);and the like. Suitable strong promoters for use in prokaryotes such asEscherichia coli include, but are not limited to Trc, Tac, T5, T7, andPLambda. Non-limiting examples of operators for use in bacterial hostcells include a lactose promoter operator (LacI repressor proteinchanges conformation when contacted with lactose, thereby preventing theLad repressor protein from binding to the operator), a tryptophanpromoter operator (when complexed with tryptophan, TrpR repressorprotein has a conformation that binds the operator; in the absence oftryptophan, the TrpR repressor protein has a conformation that does notbind to the operator), and a tac promoter operator (see, e.g., deBoer etal., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

Other examples of suitable promoters include the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to a simian virus 40 (SV40) early promoter, a mouse mammarytumor virus (MMTV) or human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, a MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the EF-1 alpha promoter, as well as human gene promoterssuch as, but not limited to, an actin promoter, a myosin promoter, ahemoglobin promoter, and a creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In some embodiments, the locus or construct or transgene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99,the disclosure of which is incorporated herein by reference). Anysuitable combination of recombinase, endonuclease, ligase, recombinationsites, etc. known to the art may be used in generating an irreversiblyswitchable promoter. Methods, mechanisms, and requirements forperforming site-specific recombination, described elsewhere herein, finduse in generating irreversibly switched promoters and are well known inthe art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006)567-605; and Tropp, Molecular Biology (2012) (Jones & BartlettPublishers, Sudbury, Mass.), the disclosures of which are incorporatedherein by reference.

In some embodiments, a nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding a TCR inducible expressioncassette. In one embodiment, the TCR inducible expression cassette isfor the production of a transgenic polypeptide product that is releasedupon TCR signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol.Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5):535-544. In some embodiments, a nucleic acid of the present disclosurefurther comprises a nucleic acid sequence encoding a cytokine operablylinked to a T-cell activation responsive promoter. In some embodiments,the cytokine operably linked to a T-cell activation responsive promoteris present on a separate nucleic acid sequence. In one embodiment, thecytokine is IL-12.

A nucleic acid of the present disclosure may be present within anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.Suitable expression vectors include, e.g., plasmids, viral vectors, andthe like. Large numbers of suitable vectors and promoters are known tothose of skill in the art; many are commercially available forgenerating a subject recombinant construct. The following vectors areprovided by way of example, and should not be construed in anyway aslimiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest.Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther.(1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995)92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. GeneTher. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum.Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus(see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); aretroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus); and thelike.

Additional expression vectors suitable for use are, e.g., withoutlimitation, a lentivirus vector, a gamma retrovirus vector, a foamyvirus vector, an adeno-associated virus vector, an adenovirus vector, apox virus vector, a herpes virus vector, an engineered hybrid virusvector, a transposon mediated vector, and the like. Viral vectortechnology is well known in the art and is described, for example, inSambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes1-4, Cold Spring Harbor Press, NY), and in other virology and molecularbiology manuals. Viruses, which are useful as vectors include, but arenot limited to, retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses.

In general, a suitable vector contains an origin of replicationfunctional in at least one organism, a promoter sequence, convenientrestriction endonuclease sites, and one or more selectable markers,(e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

In some embodiments, an expression vector (e.g., a lentiviral vector)may be used to introduce the TCR and/or the switch receptor into animmune cell or precursor thereof (e.g., a T cell). Accordingly, anexpression vector (e.g., a lentiviral vector) of the present inventionmay comprise a nucleic acid encoding for a TCR and/or a switch receptor.In some embodiments, the expression vector (e.g., lentiviral vector)will comprise additional elements that will aid in the functionalexpression of the TCR and/or switch receptor encoded therein. In someembodiments, an expression vector comprising a nucleic acid encoding fora TCR and/or a switch receptor further comprises a mammalian promoter.In one embodiment, the vector further comprises anelongation-factor-1-alpha promoter (EF-1α promoter). Use of an EF-1αpromoter may increase the efficiency in expression of downstreamtransgenes (e.g., a TCR and/or a switch receptor encoding nucleic acidsequence). Physiologic promoters (e.g., an EF-1α promoter) may be lesslikely to induce integration mediated genotoxicity, and may abrogate theability of the retroviral vector to transform stem cells. Otherphysiological promoters suitable for use in a vector (e.g., lentiviralvector) are known to those of skill in the art and may be incorporatedinto a vector of the present invention. In some embodiments, the vector(e.g., lentiviral vector) further comprises a non-requisite cis actingsequence that may improve titers and gene expression. One non-limitingexample of a non-requisite cis acting sequence is the central polypurinetract and central termination sequence (cPPT/CTS) which is important forefficient reverse transcription and nuclear import. Other non-requisitecis acting sequences are known to those of skill in the art and may beincorporated into a vector (e.g., lentiviral vector) of the presentinvention. In some embodiments, the vector further comprises aposttranscriptional regulatory element. Posttranscriptional regulatoryelements may improve RNA translation, improve transgene expression andstabilize RNA transcripts. One example of a posttranscriptionalregulatory element is the woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE). Accordingly, in some embodiments a vector forthe present invention further comprises a WPRE sequence. Variousposttranscriptional regulator elements are known to those of skill inthe art and may be incorporated into a vector (e.g., lentiviral vector)of the present invention. A vector of the present invention may furthercomprise additional elements such as a rev response element (RRE) forRNA transport, packaging sequences, and 5′ and 3′ long terminal repeats(LTRs). The term “long terminal repeat” or “LTR” refers to domains ofbase pairs located at the ends of retroviral DNAs which comprise U3, Rand U5 regions. LTRs generally provide functions required for theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. In oneembodiment, a vector (e.g., lentiviral vector) of the present inventionincludes a 3′ U3 deleted LTR. Accordingly, a vector (e.g., lentiviralvector) of the present invention may comprise any combination of theelements described herein to enhance the efficiency of functionalexpression of transgenes. For example, a vector (e.g., lentiviralvector) of the present invention may comprise a WPRE sequence, cPPTsequence, RRE sequence, 5′LTR, 3′ U3 deleted LTR′ in addition to anucleic acid encoding for a TCR and/or a switch receptor.

Vectors of the present invention may be self-inactivating vectors. Asused herein, the term “self-inactivating vector” refers to vectors inwhich the 3′ LTR enhancer promoter region (U3 region) has been modified(e.g., by deletion or substitution). A self-inactivating vector mayprevent viral transcription beyond the first round of viral replication.Consequently, a self-inactivating vector may be capable of infecting andthen integrating into a host genome (e.g., a mammalian genome) onlyonce, and cannot be passed further. Accordingly, self-inactivatingvectors may greatly reduce the risk of creating a replication-competentvirus.

In some embodiments, a nucleic acid of the present invention may be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known to those of skill in the art; any known method can be used tosynthesize RNA comprising a sequence encoding a TCR and/or switchreceptor of the present disclosure. Methods for introducing RNA into ahost cell are known in the art. See, e.g., Zhao et al. Cancer Res.(2010) 15: 9053. Introducing RNA comprising a nucleotide sequenceencoding a TCR and/or switch receptor of the present disclosure into ahost cell can be carried out in vitro or ex vivo or in vivo. Forexample, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.)can be electroporated in vitro or ex vivo with RNA comprising anucleotide sequence encoding a TCR and/or switch receptor of the presentdisclosure.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell may also containeither a selectable marker gene or a reporter gene, or both, tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In some embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, without limitation, antibiotic-resistancegenes.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity.

Expression of the reporter gene is assessed at a suitable time after theDNA has been introduced into the recipient cells. Suitable reportergenes may include, without limitation, genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

E. Modified Immune Cells

The present disclosure provides a modified immune cell or precursorthereof (e.g., a T cell) comprising an exogenous TCR and/or switchreceptor as described herein. Accordingly, such modified cells possessthe specificity directed by the TCR that is expressed therein. Forexample, a modified cell of the present disclosure comprising a NY-ESO-1TCR possesses specificity for NY-ESO-1 on a target cell.

In some embodiments, a modified cell of the present disclosure comprisesan exogenous TCR. In one embodiment, a modified cell of the presentdisclosure comprises an exogenous TCR having affinity for NY-ESO-1 on atarget cell. In some embodiments, a modified cell of the presentdisclosure comprises an exogenous TCR and a switch receptor. In oneembodiment, a modified cell of the present disclosure comprises anexogenous TCR having affinity for NY-ESO-1 on a target cell, and aswitch receptor. Modified cells comprising a switch receptor of thepresent disclosure are able to activate inhibitory ligands in themicroenvironment by virtue of the first domain of the switch receptor,and switch the otherwise inhibitory signal into a positive signal to themodified cell by way of signaling through the second domain of theswitch receptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a PD1-CD28 switch receptor. Such modified cells(e.g., modified T cells) in addition to having affinity for NY-ESO-1 ona target cell, are capable of converting inhibitory signals from themicroenvironment they reside in into activating signals in the T cell.Modified T cells comprising a PD1-CD28 switch receptor are capable ofconverting inhibitory PD-1 ligands in the microenvironment intoactivating signals by signaling through the CD28 domain of the switchreceptor. As such, a modified cell comprising an exogenous TCR (havingaffinity for an antigen on a target cell) and a switch receptor of thepresent disclosure possesses specificity for the antigen on the targetcell, and can bypass any inhibitory, immunosuppressive signals presentin the microenvironment wherein the target cell resides. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a PD1-CD28 switchreceptor capable of converting an inhibitory, immunosuppressive PD-1ligand in the tumor microenvironment into an activating signal in themodified cell (resulting in an activated modified cell). As used herein,an “activated modified cell” or “activated modified T cell” refers to,among other things, modified cells that are undergoing cell division.Activation can also be associated with generating an immune response,and detectably unregulating surface markers. Upregulation of surfacemarkers, such as CD25 (the IL-2 receptor), initiates a phosphorylationcascade involving p561ck, causes the release of cytokines andinterleukins, increases DNA synthesis, and causes the cells toproliferate.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a TIM3-CD28 switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a TIM3-CD28 switchreceptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a PD1-41BB switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a PD1-41 BB switchreceptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a PD1^(A132L)-41BB switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a PD1^(A132L)-41BBswitch receptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a PD1^(A132L)-CD28 switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a PD1^(A132L)-CD28switch receptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a TGFβRI-IL-12Rβ1 switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a TGFβRI-IL-12Rβ1switch receptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a TGFβRII-IL-12Rβ2 switch receptor. In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a TGFβRII-IL-12Rβ2switch receptor.

In an exemplary embodiment, a modified cell of the present disclosurecomprises an exogenous TCR (e.g., a TCR having affinity for NY-ESO-1 ona target cell), and a TGFβRIIDN “switch receptor.” In an exemplaryembodiment, a modified cell (e.g., T cell) comprises an exogenous TCRhaving affinity for NY-ESO-1 on a tumor cell, and a TGFβRIIDN “switchreceptor.”

Gene Edited Immune Cells

The present disclosure provides gene edited modified cells. In someembodiments, a modified cell (e.g., a modified cell comprising anexogenous TCR and/or switch receptor) of the present disclosure isgenetically edited to disrupt the expression of one or more endogenouslyexpressed genes. In some embodiments, the gene-edited immune cells(e.g., T cells), having a reduction, deletion, elimination, knockout ordisruption in expression of an endogenous receptor (e.g. an endogenous Tcell receptor or immune checkpoint protein).

In certain embodiments, the modified cell of the present disclosure isgenetically edited to disrupt the expression of endogenous TCR geneproducts (e.g., gene products of TRAC and TRBC). Without being bound toany theory, disrupting the expression of TRAC and/or TRBC results in 1)reduced endogenous TCR and exogenous TCR (e.g., an NY-ESO-1 TCR)mispairing, thus reducing the risk of autoreactivity; and 2) enhancesexogenous TCR expression on the cell surface by reducing mispairing withendogenous TCR, thus increasing efficacy of the modified cells. In oneembodiment, the modified cell of the present disclosure is geneticallyedited to disrupt the expression of endogenous PDCD1 gene products(Programmed Death 1 receptor; PD-1). Disrupting the expression ofendogenous PD-1 may create “checkpoint” resistant modified cells,resulting in increased tumor control. Checkpoint resistant modifiedcells may also be created by disrupting the expression of, for example,without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276),B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA/CD272),CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4/CD152),Indoleamine 2,3-dioxygenase (IDO), the Killer-cell Immunoglobulin-likeReceptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cellimmunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulindomain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of Tcell activation (VISTA). Accordingly, in one embodiment, the modifiedcell of the present disclosure is genetically edited to disrupt theexpression of endogenous CTLA-4.

In some embodiments, a modified cell of the present disclosure comprisesan exogenous TCR and is genetically edited to disrupt the expression ofone or more endogenously expressed genes. In one embodiment, a modifiedcell of the present disclosure comprises an exogenous TCR havingaffinity for NY-ESO-1 on a target cell, wherein the expression of one ormore endogenous genes is downregulated. In one embodiment, a modifiedcell of the present disclosure is a modified T cell comprising anexogenous TCR having affinity for NY-ESO-1 on a target cell, wherein theexpression of one or more of A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA(CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and/orVISTA gene products are downregulated. In an exemplary embodiment, amodified cell of the present disclosure is a modified T cell comprisingan exogenous TCR having affinity for NY-ESO-1 on a target cell, whereinthe expression of TRAC, TRBC, and PDCD1 gene products are downregulated.

In some embodiments, a modified cell of the present disclosure comprisesan exogenous TCR and a switch receptor, and is genetically edited todisrupt the expression of one or more endogenously expressed genes. Inone embodiment, a modified cell of the present disclosure comprises, anexogenous TCR having affinity for NY-ESO-1 on a target cell, and aswitch receptor, wherein the expression of one or more endogenous genesis downregulated. In one embodiment, a modified cell of the presentdisclosure is a modified T cell comprising an exogenous TCR havingaffinity for NY-ESO-1 on a target cell, and a switch receptor, whereinthe expression of one or more of A2AR, B7-H3 (CD276), B7-H4 (VTCN1),BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and/orVISTA gene products are downregulated. In one embodiment, a modifiedcell of the present disclosure is a modified T cell comprising anexogenous TCR having affinity for NY-ESO-1 on a target cell, and aswitch receptor, wherein the expression of TRAC and TRBC gene productsare downregulated. In an exemplary embodiment, a modified cell of thepresent disclosure is a modified T cell comprising an exogenous TCRhaving affinity for NY-ESO-1 on a target cell, and a PD1-CD28 switchreceptor, wherein the expression of TRAC and TRBC gene products aredownregulated. In an exemplary embodiment, a modified cell of thepresent disclosure is a modified T cell comprising an exogenous TCRhaving affinity for NY-ESO-1 on a target cell, and a PD1-CD28 switchreceptor, wherein the expression of TRAC, TRBC and TIM-3 gene productsare downregulated.

A modified cell of the present disclosure can comprise various switchreceptors described elsewhere herein, e.g., PD1-CD28, TIM3-CD28,PD1-41BB, PD1^(A132L)-41BB, PD1^(A132L)-CD28, TGFβRI-IL-12Rβ1,TGFβRII-IL-12Rβ2, TGFβRIIDN.

Various gene editing technologies are known to those skilled in the art.Gene editing technologies include, without limitation, homingendonucleases, zinc-finger nucleases (ZFNs), transcriptionactivator-like effector (TALE) nucleases (TALENs), and clusteredregularly interspaced short palindromic repeats (CRISPR) systems (e.g.CRISPR/CRISPR-associated protein 9 (Cas9)). Homing endonucleasesgenerally cleave their DNA substrates as dimers, and do not havedistinct binding and cleavage domains. ZFNs recognize target sites thatconsist of two zinc-finger binding sites that flank a 5- to 7-base pair(bp) spacer sequence recognized by the FokI cleavage domain. TALENsrecognize target sites that consist of two TALE DNA-binding sites thatflank a 12- to 20-bp spacer sequence recognized by the FokI cleavagedomain. The Cas9 nuclease is targeted to DNA sequences complementary tothe targeting sequence within the single guide RNA (gRNA) locatedimmediately upstream of a compatible protospacer adjacent motif (PAM).Accordingly, one of skill in the art would be able to select theappropriate gene editing technology for the present invention.

In some aspects, the disruption is carried out by gene editing using anRNA-guided nuclease such as a clustered regularly interspersed shortpalindromic nucleic acid (CRISPR)-Cas system, such as CRISPR/Cas9system, specific for the gene (e.g., TRAC, TRBC, PDCDI or TIM3) beingdisrupted. In some embodiments, an agent containing a Cas9 (e.g. Cas9RNA) and a guide RNA (gRNA) containing a targeting domain, which targetsa region of the genetic locus, is introduced into the cell. In someembodiments, the agent is or comprises a ribonucleoprotein (RNP) complexof Cas9 and gRNA containing the gene-targeted targeting domain(Cas9/gRNA RNP). In some embodiments, the introduction includescontacting the agent or portion thereof with the cells, in vitro, whichcan include cultivating or incubating the cell and agent for up to 24,36 or 48 hours or 3, 4, 5, 6, 7, or 8 days. In some embodiments, theintroduction further can include effecting delivery of the agent intothe cells. In various embodiments, the methods, compositions and cellsaccording to the present disclosure utilize direct delivery ofribonucleoprotein (RNP) complexes of Cas9 and gRNA to cells, for exampleby electroporation. In some embodiments, the RNP complexes include agRNA that has been modified to include a 3′ poly-A tail and a 5′Anti-Reverse Cap Analog (ARCA) cap.

The CRISPR/Cas9 system is a facile and efficient system for inducingtargeted genetic alterations. Target recognition by the Cas9 proteinrequires a ‘seed’ sequence within the guide RNA (gRNA) and a conserveddi-nucleotide containing protospacer adjacent motif (PAM) sequenceupstream of the gRNA-binding region. The CRISPR/Cas9 system can therebybe engineered to cleave virtually any DNA sequence by redesigning thegRNA in cell lines (such as 293T cells), primary cells, and TCR T cells.The CRISPR/Cas9 system can simultaneously target multiple genomic lociby co-expressing a single Cas9 protein with two or more gRNAs, makingthis system suited for multiple gene editing or synergistic activationof target genes.

The Cas9 protein and guide RNA form a complex that identifies andcleaves target sequences. Cas9 is comprised of six domains: REC I, RECII, Bridge Helix, PAM interacting, HNH, and RuvC. The RecI domain bindsthe guide RNA, while the Bridge helix binds to target DNA. The HNH andRuvC domains are nuclease domains. Guide RNA is engineered to have a 5′end that is complementary to the target DNA sequence. Upon binding ofthe guide RNA to the Cas9 protein, a conformational change occursactivating the protein. Once activated, Cas9 searches for target DNA bybinding to sequences that match its protospacer adjacent motif (PAM)sequence. A PAM is a two or three nucleotide base sequence within onenucleotide downstream of the region complementary to the guide RNA. Inone non-limiting example, the PAM sequence is 5′-NGG-3′. When the Cas9protein finds its target sequence with the appropriate PAM, it melts thebases upstream of the PAM and pairs them with the complementary regionon the guide RNA. Then the RuvC and HNH nuclease domains cut the targetDNA after the third nucleotide base upstream of the PAM.

One non-limiting example of a CRISPR/Cas system used to inhibit geneexpression, CRISPRi, is described in U.S. Patent Appl. Publ. No.US20140068797. CRISPRi induces permanent gene disruption that utilizesthe RNA-guided Cas9 endonuclease to introduce DNA double stranded breakswhich trigger error-prone repair pathways to result in frame shiftmutations. A catalytically dead Cas9 lacks endonuclease activity. Whencoexpressed with a guide RNA, a DNA recognition complex is generatedthat specifically interferes with transcriptional elongation, RNApolymerase binding, or transcription factor binding. This CRISPRi systemefficiently represses expression of targeted genes.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequencespecific for a target gene and a Cas endonuclease are introduced into acell and form a complex that enables the Cas endonuclease to introduce adouble strand break at the target gene. In certain embodiments, theCRISPR/Cas system comprises an expression vector, such as, but notlimited to, an pAd5F35-CRISPR vector. In other embodiments, the Casexpression vector induces expression of Cas9 endonuclease. Otherendonucleases may also be used, including but not limited to, T7, Cas3,Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1,other nucleases known in the art, and any combinations thereof.

In certain embodiments, inducing the Cas expression vector comprisesexposing the cell to an agent that activates an inducible promoter inthe Cas expression vector. In such embodiments, the Cas expressionvector includes an inducible promoter, such as one that is inducible byexposure to an antibiotic (e.g., by tetracycline or a derivative oftetracycline, for example doxycycline). Other inducible promoters knownby those of skill in the art can also be used. The inducing agent can bea selective condition (e.g., exposure to an agent, for example anantibiotic) that results in induction of the inducible promoter. Thisresults in expression of the Cas expression vector.

The guide RNA is specific for a genomic region of interest and targetsthat region for Cas endonuclease-induced double strand breaks. Thetarget sequence of the guide RNA sequence may be within a locus of agene or within a non-coding region of the genome. In certainembodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

Guide RNA (gRNA), also referred to as “short guide RNA” or “sgRNA”,provides both targeting specificity and scaffolding/binding ability forthe Cas9 nuclease. The gRNA can be a synthetic RNA composed of atargeting sequence and scaffold sequence derived from endogenousbacterial crRNA and tracrRNA. gRNA is used to target Cas9 to a specificgenomic locus in genome engineering experiments. Guide RNAs can bedesigned using standard tools well known in the art.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to have somecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In certain embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In otherembodiments, the target sequence may be within an organelle of aeukaryotic cell, for example, mitochondrion or nucleus. Typically, inthe context of an endogenous CRISPR system, formation of a CRISPRcomplex (comprising a guide sequence hybridized to a target sequence andcomplexed with one or more Cas proteins) results in cleavage of one orboth strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 50 or more base pairs) the target sequence. As with the targetsequence, it is believed that complete complementarity is not needed,provided this is sufficient to be functional.

In certain embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a host cell, suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Cas enzyme,a guide sequence linked to a tracr-mate sequence, and a tracr sequencecould each be operably linked to separate regulatory elements onseparate vectors. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements may be combined in asingle vector, with one or more additional vectors providing anycomponents of the CRISPR system not included in the first vector. CRISPRsystem elements that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a secondelement. The coding sequence of one element may be located on the sameor opposite strand of the coding sequence of a second element, andoriented in the same or opposite direction. In certain embodiments, asingle promoter drives expression of a transcript encoding a CRISPRenzyme and one or more of the guide sequence, tracr mate sequence(optionally operably linked to the guide sequence), and a tracr sequenceembedded within one or more intron sequences (e.g., each in a differentintron, two or more in at least one intron, or all in a single intron).

In certain embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Additional domains that mayform part of a fusion protein comprising a CRISPR enzyme are describedin U.S. Patent Appl. Publ. No. US20110059502, incorporated herein byreference. In certain embodiments, a tagged CRISPR enzyme is used toidentify the location of a target sequence.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian and non-mammalian cells ortarget tissues. Such methods can be used to administer nucleic acidsencoding components of a CRISPR system to cells in culture, or in a hostorganism. Non-viral vector delivery systems include DNA plasmids, RNA(e.g., a transcript of a vector described herein), naked nucleic acid,and nucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell(Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy1:13-26).

In some embodiments, the CRISPR/Cas is derived from a type II CRISPR/Cassystem. In other embodiments, the CRISPR/Cas system is derived from aCas9 protein. The Cas9 protein can be from Streptococcus pyogenes,Streptococcus thermophilus, or other species.

In general, Cas proteins comprise at least one RNA recognition and/orRNA binding domain. RNA recognition and/or RNA binding domains interactwith the guiding RNA. Cas proteins can also comprise nuclease domains(i.e., DNase or RNase domains), DNA binding domains, helicase domains,RNAse domains, protein-protein interaction domains, dimerizationdomains, as well as other domains. The Cas proteins can be modified toincrease nucleic acid binding affinity and/or specificity, alter anenzymatic activity, and/or change another property of the protein. Incertain embodiments, the Cas-like protein of the fusion protein can bederived from a wild type Cas9 protein or fragment thereof. In otherembodiments, the Cas can be derived from modified Cas9 protein. Forexample, the amino acid sequence of the Cas9 protein can be modified toalter one or more properties (e.g., nuclease activity, affinity,stability, and so forth) of the protein. Alternatively, domains of theCas9 protein not involved in RNA-guided cleavage can be eliminated fromthe protein such that the modified Cas9 protein is smaller than the wildtype Cas9 protein. In general, a Cas9 protein comprises at least twonuclease (i.e., DNase) domains. For example, a Cas9 protein can comprisea RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC andHNH domains work together to cut single strands to make adouble-stranded break in DNA. (Jinek, et al., 2012, Science,337:816-821). In certain embodiments, the Cas9-derived protein can bemodified to contain only one functional nuclease domain (either aRuvC-like or a HNH-like nuclease domain). For example, the Cas9-derivedprotein can be modified such that one of the nuclease domains is deletedor mutated such that it is no longer functional (i.e., the nucleaseactivity is absent). In some embodiments in which one of the nucleasedomains is inactive, the Cas9-derived protein is able to introduce anick into a double-stranded nucleic acid (such protein is termed a“nickase”), but not cleave the double-stranded DNA. In any of theabove-described embodiments, any or all of the nuclease domains can beinactivated by one or more deletion mutations, insertion mutations,and/or substitution mutations using well-known methods, such assite-directed mutagenesis, PCR-mediated mutagenesis, and total genesynthesis, as well as other methods known in the art.

In one non-limiting embodiment, a vector drives the expression of theCRISPR system. The art is replete with suitable vectors that are usefulin the present invention. The vectors to be used are suitable forreplication and, optionally, integration in eukaryotic cells. Typicalvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence. The vectors of the present invention mayalso be used for nucleic acid standard gene delivery protocols. Methodsfor gene delivery are known in the art (U.S. Pat. Nos. 5,399,346,5,580,859 & 5,589,466, incorporated by reference herein in theirentireties).

Further, the vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (4th Edition, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,2012), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitablevector contains an origin of replication functional in at least oneorganism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers (e.g., WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

In some embodiments, guide RNA(s) and Cas9 can be delivered to a cell asa ribonucleoprotein (RNP) complex (e.g., a Cas9/RNA-protein complex).RNPs are comprised of purified Cas9 protein complexed with gRNA and arewell known in the art to be efficiently delivered to multiple types ofcells, including but not limited to stem cells and immune cells(Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI). In someembodiments, the Cas9/RNA-protein complex is delivered into a cell byelectroporation.

In some embodiments, a gene edited modified cell of the presentdisclosure is edited using CRISPR/Cas9 to disrupt one or more endogenousgenes in a modified cell (e.g., a modified T cell). In some embodiments,CRISPR/Cas9 is used to disrupt one or more of endogenous TRAC, TRBC,PDCD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4(CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and/or VISTA loci, therebyresulting in the downregulation of TRAC, TRBC, PD-1, A2AR, B7-H3(CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR,LAG3, TIGIT, TIM-3, and/or VISTA. In some embodiments, CRISPR/Cas9 isused to disrupt one or more of endogenous TRAC, TRBC, PDCD1, and/orTIM-3. Suitable gRNAs for use in disrupting one or more of endogenousTRAC, TRBC, PDCD1, and/or TIM-3 is set forth in Table 1.

TABLE 1 SEQ ID gRNA name gRNA sequence NO: TRAC1-1 GAGAATCAAAATCGGTGAAT 37 TRAC1-4 TGTGCTAGACATGAGGTCTA  38 TRAC1-5 AAAGTCAGATTTGTTGCTCC  39TRAC1-9 AGAGTCTCTCAGCTGGTACA  40 TRAC1-13 AGCTGGTACACGGCAGGGTC  41TRAC1-16 ACAAAACTGTGCTAGACATG  42 TRAC2-1 CTCGACCAGCTTGACATCAC  43TRAC2-2 AAGTTCCTGTGATGTCAAGC  44 TRAC3-1 TTCGGAACCCAATCACTGAC  45TRAC3-2 TTAATCTGCTCATGACGCTG  46 TRAC3-3 GATTAAACCCGGCCACTTTC  47TRAC3-4 CGTCATGAGCAGATTAAACC  48 TRAC3-5 TAAACCCGGCCACTTTCAGG  49TRBC11-1 CAAACACAGCGACCTCGGGT  50 TRBC11-2 GGCTCAAACACAGCGACCTC  51TRBC11-3 TCAAACACAGCGACCTCGGG  52 TRBC11-4 TGGCTCAAACACAGCGACCT  53TRBC12-1 TCTCCGAGAGCCCGTAGAAC  54 TRBC12-2 GGCTCTCGGAGAATGACGAG  55TRBC12-3 TGACAGCGGAAGTGGTTGCG  56 TRBC12-4 AGTCCAGTTCTACGGGCTCT  57TRBC12-5 CGCTGTCAAGTCCAGTTCTA  58 TRBC12-6 AGCTCAGCTCCACGTGGTCG  59TRBC12-7 ACTGGACTTGACAGCGGAAG  60 TRBC12-8 TTGACAGCGGAAGTGGTTGC  61TRBC12-9 GACAGCGGAAGTGGTTGCGG  62 TRBC12-10 TGACGAGTGGACCCAGGATA  63TRBC12-11 CGTAGAACTGGACTTGACAG  64 TRBC12-12 ATGACGAGTGGACCCAGGAT  65TRBC12-13 CTTGACAGCGGAAGTGGTTG  66 TRBC12-14 GCTGTCAAGTCCAGTTCTAC  67TRBC13-1 AGGCCTCGGCGCTGACGATC  68 TRBC13-2 GGCCTCGGCGCTGACGATCT  69TRBC13-3 CACCCAGATCGTCAGCGCCG  70 TRBC13-4 GACGATCTGGGTGACGGGTT  71TRBC13-5 GATCGTCAGCGCCGAGGCCT  72 TRBC13-6 AGATCGTCAGCGCCGAGGCC  73PD1.1-1 TGTAGCACCGCCCAGACGAC  74 PD1.1-2 CGTCTGGGCGGTGCTACAAC  75PD1.1-3 GTCTGGGCGGTGCTACAACT  76 PD1.1-4 AGGCGCCCTGGCCAGTCGTC  77PD1.1-5 CACCGCCCAGACGACTGGCC  78 PD1.21-1 ATGTGGAAGTCACGCCCGTT  79PD1.21-2 CATGTGGAAGTCACGCCCGT  80 PD1.21-3 CACGAAGCTCTCCGATGTGT  81PD1.21-4 CGGAGAGCTTCGTGCTAAAC  82 PD1.21-5 CCTGCTCGTGGTGACCGAAG  83PD1.21-6 CCCCTTCGGTCACCACGAGC  84 PD1.21-7 AGGCGGCCAGCTTGTCCGTC  85PD1.21-8 GCCCTGCTCGTGGTGACCGA  86 PD1.21-9 CCCTTCGGTCACCACGAGCA  87PD1.21-10 CCCTGCTCGTGGTGACCGAA  88 PD1.22-1 GCGTGACTTCCACATGAGCG  89PD1.22-2 AGGTGCCGCTGTCATTGCGC  90 PD1.22-3 ACTTCCACATGAGCGTGGTC  91PD1.22-4 GGTGCCGCTGTCATTGCGCC  92 PD1.3-1 ACCCTGGTGGTTGGTGTCGT  93PD1.3-2 AGGGTTTGGAACTGGCCGGC  94 PD1.5-1 ATTGTCTTTCCTAGCGGAAT  95PD1.5-2 TCAGTGGCTGGGCACTCCGA  96 PD1.5-3 CATTGTCTTTCCTAGCGGAA  97 Tim3-1AATGTGACTCTAGCAGACAG  98 Tim3-2 ATGAGAATACCCTAGTAAGG  99 Tim3-3TATGAGAATACCCTAGTAAG 100 Tim3-4 TGGCCCAGGTAACTATGCAT 101 Tim3-5ATAGGCATCTACATCGGAGC 102 Tim3-6 GCTGTGGAAATAAAGTGTTG 103 Tim3-7GTGGAATACAGAGCGGAGGT 104 Tim3-8 ACAGTGGGATCTACTGCTGC 105 Tim3-9TCTCTCTGCCGAGTCGGTGC 106 Tim3-10 TTATGCCTGGGATTTGGATC 107 Tim3-11ATCAGAATAGGCATCTACAT 108 Tim3-12 TGAGTTACGGGACTCTAGAT 109 Tim3-13GCCAATGTGGATATTTGCTA 110 Tim3-14 GTGAAGTCTCTCTGCCGAGT 111 Tim3-15TCAGGGACACATCTCCTTTG 112 Tim3-16 GGGCACGAGGTTCCCTGGGG 113 Tim3-17AAATAAGGTGGTTGGATCTA 114 Tim3-18 CTAAATGGGGATTTCCGCAA 115 Tim3-19AATGTGGCAACGTGGTGCTC 116 Tim3-20 ATCCCCATTTAGCCAGTATC 117 Tim3-21TGCTGCCGGATCCAAATCCC 118 Tim3-22 GAACCTCGTGCCCGTCTGCT 119 Tim3-23CAGACGGGCACGAGGTTCCC 120 Tim3-24 AGACGGGCACGAGGTTCCCT 121 Tim3-25CTCTCTGCCGAGTCGGTGCA 122 Tim3-26 TCTCTGCCGAGTCGGTGCAG 123 Tim3-27AGGTCACCCCTGCACCGACT 124 Tim3-28 TAGGCATCTACATCGGAGCA 125 Tim3-29TAGATTGGCCAATGACTTAC 126

Suitable gRNAs for use in disrupting one or more of endogenous TRAC,TROC, and/or PDCD1 are set forth in Table 2 as Group I gRNAs and GroupII gRNAs. In an exemplary embodiment, gene edited modified cells of thepresent disclosure are edited using any one of the Group I gRNAstargeted to TRAC, TRC, and PD1. In an exemplary embodiment, genemodified cells of the present disclosure are edited using any one of theGroup II gRNAs targeted to TRAC, TRBC, and PD1.

TABLE 2 SEQ ID gRNA name gRNA sequence NO: TRAC-Group 1TGTGCTAGACATGAGGTCTA  38 TRAC-Group 2 CGTCATGAGCAGATTAAACC  48TRBC-Group 1 GGAGAATGACGAGTGGACCC 131 TRBC-Group 2 ATGACGAGTGGACCCAGGAT 65 PD1-Group 1 GGCGCCCTGGCCAGTCGTCT 127 PD1-Group 2GTCTGGGCGGTGCTACAACT  76

Accordingly, a method of genetically editing a modified cell of thepresent disclosure comprises introducing into the cell one or morenucleic acids capable of downregulating gene expression of one or moreendogenous genes selected from TRAC, TRBC, PDCD1, A2AR, B7-H3 (CD276),B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3,TIGIT, TIM-3, and VISTA. In one embodiment, a method of geneticallyediting a modified cell of the present disclosure comprises introducinginto the cell one or more nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from TRAC, TRBC,PDCD1, and TIM-3. In one embodiment, a method for generating a modifiedcell of the present disclosure comprises 1) introducing into the cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR; and 2) introducing into the cell one or more nucleic acids capableof downregulating gene expression of one or more endogenous genesselected from TRAC, TRBC, PDCD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1),BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR, LAG3, TIGIT, TIM-3, andVISTA. In one embodiment, a method for generating a modified cell of thepresent disclosure comprises 1) introducing into the cell a nucleic acidcomprising a nucleic acid sequence encoding an exogenous TCR; and 2)introducing into the cell one or more nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom TRAC, TRBC, PDCD1, and TIM-3. In an exemplary embodiment, a methodfor generating a modified T cell of the present disclosure comprises 1)introducing into the T cell a nucleic acid comprising a nucleic acidsequence encoding an exogenous TCR having affinity for NY-ESO-1 on atarget cell; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of TRAC (e.g., SEQ ID NOs:37-49); 3)introducing into the cell a nucleic acid capable of downregulating geneexpression of TRBC (e.g., SEQ ID NOs:50-73, or 131); and 4) introducinginto the cell a nucleic acid capable of downregulating gene expressionof PDCD1 (e.g., SEQ ID NOs:74-97, or 127). In an exemplary embodiment, amethod for generating a modified T cell of the present disclosurecomprises 1) introducing into the T cell a nucleic acid comprising anucleic acid sequence encoding an exogenous TCR having affinity forNY-ESO-1 on a target cell; 2) introducing into the cell a nucleic acidcapable of downregulating gene expression of TRAC (e.g., SEQ IDNOs:37-49); 3) introducing into the cell a nucleic acid capable ofdownregulating gene expression of TRBC (e.g., SEQ ID NOs:50-73, or 131);and 4) introducing into the cell a nucleic acid capable ofdownregulating gene expression of TIM-3 (e.g., SEQ ID NOs:98-126).

In one embodiment, a method for generating a modified cell of thepresent disclosure comprises 1) introducing into the cell a nucleic acidcomprising a nucleic acid sequence encoding an exogenous TCR and anucleic acid sequence encoding a switch receptor; and 2) introducinginto the cell one or more nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from TRAC, TRBC,PDCD1, and TIM-3. In an exemplary embodiment, a method for generating amodified T cell of the present disclosure comprises 1) introducing intothe T cell a nucleic acid comprising a nucleic acid sequence encoding anexogenous TCR having affinity for NY-ESO-1 on a target cell, and anucleic acid sequence encoding a PD1-CD28 switch receptor; 2)introducing into the cell a nucleic acid capable of downregulating geneexpression of TRAC (e.g., SEQ ID NOs:37-49); and 3) introducing into thecell a nucleic acid capable of downregulating gene expression of TRBC(e.g., SEQ ID NOs:50-73, or 131). In an exemplary embodiment, a methodfor generating a modified T cell of the present disclosure comprises 1)introducing into the T cell a nucleic acid comprising a nucleic acidsequence encoding an exogenous TCR having affinity for NY-ESO-1 on atarget cell, and a nucleic acid sequence encoding a PD1-CD28 switchreceptor; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of TRAC (e.g., SEQ ID NOs:37-49); 3)introducing into the cell a nucleic acid capable of downregulating geneexpression of TRBC (e.g., SEQ ID NOs:50-73, or 131); and 4) introducinginto the cell a nucleic acid capable of downregulating gene expressionof TIM-3 (e.g., SEQ ID NOs:98-126).

In some embodiments, a gene edited modified cell of the presentdisclosure comprises at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in the endogenous TIM-3 codingsequence. In some embodiments, a gene edited modified cell of thepresent disclosure is edited by any one of the TIM-3-targeted nucleicacids described herein, e.g., SEQ ID NOs:98-126.

In some embodiments, a gene edited modified cell of the presentdisclosure comprises at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in the endogenous TCR alpha chaincoding sequence (TRAC). In some embodiments, a gene edited modified cellof the present disclosure is edited by any one of the TRAC-targetednucleic acids described herein, e.g., SEQ ID NOs:37-49. In someembodiments, a gene edited modified cell of the present disclosure isedited by a Group I or Group II TRAC-targeted nucleic acid, as describedherein. In an exemplary embodiment, a gene edited modified cell of thepresent disclosure is edited by a Group I TRAC-targeted nucleic acid,and comprises at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion in the endogenous TCR alpha chain codingsequence (TRAC) comprising the nucleic acid sequence:AACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCT (SEQ ID NO:128), resulting indownregulation of the expression of endogenous TCR alpha chain. Forexample, a gene edited modified cell of the present disclosure is editedby a Group I TRAC-targeted nucleic acid and may comprise any one of theedited endogenous TRAC coding sequences as shown in FIG. 1 .

In some embodiments, a gene edited modified cell of the presentdisclosure comprises at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in the endogenous TCR beta chaincoding sequence (TRBC). In some embodiments, a gene edited modified cellof the present disclosure is edited by any one of the TRBC-targetednucleic acids described herein, e.g., SEQ ID NOs:50-73, or 131. In someembodiments, a gene edited modified cell of the present disclosure isedited by a Group I or Group II TRAC-targeted nucleic acid, as describedherein. In an exemplary embodiment, a gene edited modified cell of thepresent disclosure is edited by a Group I TRBC-targeted nucleic acid,and comprises at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion in the endogenous TCR beta coding sequence(TRBC) comprising the nucleic acid sequence:GCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCG (SEQ ID NO:129), resulting indownregulation of the expression of endogenous TCR beta chain. Forexample, a gene edited modified cell of the present disclosure is editedby a Group I TRBC-targeted nucleic acid and may comprise any one of theedited endogenous TRBC coding sequences as shown in FIG. 2 .

In some embodiments, a gene edited modified cell of the presentdisclosure comprises at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in the endogenous PD1 codingsequence. In some embodiments, a gene edited modified cell of thepresent disclosure is edited by any one of the PD1-targeted nucleicacids described herein, e.g., SEQ ID NOs:74-97, or 127. In someembodiments, a gene edited modified cell of the present disclosure isedited by a Group I or Group II PD1-targeted nucleic acid, as describedherein. In an exemplary embodiment, a gene edited modified cell of thepresent disclosure is edited by a Group I PD1-targeted nucleic acid, andcomprises at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion in the PD1 coding sequence comprising thenucleic acid sequence:

(SEQ ID NO: 130) GCTGCTCCAGGCATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGGTAGGTGGGGTC G,resulting in downregulation of the expression of endogenous PD1. Forexample, a gene edited modified cell of the present disclosure is editedby a Group I PD1-targeted nucleic acid and may comprise any one of theedited endogenous PD1 coding sequences as shown in FIG. 3 .

In some embodiments, a gene edited modified cell of the presentdisclosure comprises at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion in the endogenous TCR alpha chaincoding sequence (TRAC) comprising the nucleic acid sequence set forth inSEQ ID NO:128, in the endogenous TCR beta chain coding sequence (TRBC)comprising the nucleic acid sequence set forth in SEQ ID NO:129, andoptionally in the endogenous PD1 coding sequence (PDCD1) comprising thenucleic acid sequence set forth in SEQ ID NO:130, resulting indownregulation of the expression of endogenous TCR alpha chain,endogenous TCR beta chain, and endogenous PD1.

In one embodiment, a modified cell of the present disclosure comprisesan exogenous TCR, wherein the expression of an endogenous TCR alphachain coding sequence, an endogenous TCR beta chain coding sequence, andan endogenous PD1 coding sequence is downregulated. In an exemplaryembodiment, a modified cell of the present disclosure is a modified Tcell comprising an exogenous TCR having affinity for NY-ESO-1 on atarget cell, wherein the expression of an endogenous TCR alpha chaincoding sequence, an endogenous TCR beta chain coding sequence, and anendogenous PD1 coding sequence are downregulated. In another exemplaryembodiment, a modified cell of the present disclosure is a modified Tcell comprising an exogenous TCR having affinity for NY-ESO-1 on atarget cell, wherein at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion is in the endogenous TCR alphachain coding sequence comprising the nucleic acid sequence set forth inSEQ ID NO:128, wherein at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion is in the endogenous TCR beta chaincoding sequence comprising the nucleic acid sequence set forth in SEQ IDNO: 129, and wherein at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion is in the endogenous PD1 codingsequence comprising the nucleic acid sequence set forth in SEQ IDNO:130, thereby resulting in the downregulation of the expression ofendogenous TCR alpha chain, endogenous TCR beta chain, and endogenousPD1.

In one embodiment, a modified cell of the present disclosure comprisesan exogenous TCR and a switch receptor (e.g., a PD1-CD28 switchreceptor), wherein the expression of an endogenous TCR alpha chaincoding sequence, and an endogenous TCR beta chain coding sequence aredownregulated. In an exemplary embodiment, a modified cell of thepresent disclosure is a modified T cell comprising an exogenous TCRhaving affinity for NY-ESO-1 on a target cell and a switch receptor(e.g., a PD1-CD28 switch receptor), wherein the expression of anendogenous TCR alpha chain coding sequence, and an endogenous TCR betachain coding sequence are downregulated. In another exemplaryembodiment, a modified cell of the present disclosure is a modified Tcell comprising an exogenous TCR having affinity for NY-ESO-1 on atarget cell and a switch receptor (e.g., a PD1-CD28 switch receptor),wherein at least one nucleotide substitution, deletion, insertion,and/or insertion/deletion is in the endogenous TCR alpha chain codingsequence comprising the nucleic acid sequence set forth in SEQ IDNO:128, and wherein at least one nucleotide substitution, deletion,insertion, and/or insertion/deletion is in the endogenous TCR beta chaincoding sequence comprising the nucleic acid sequence set forth in SEQ IDNO:129, thereby resulting in the downregulation of the expression ofendogenous TCR alpha chain and endogenous TCR beta chain.

In some aspects, the provided compositions and methods include those inwhich at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95% of immune cells in a composition of immune cells contain thedesired genetic modification. For example, about 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% or 95% of immune cells in a composition of cells intowhich an agent (e.g. gRNA/Cas9) for knockout or genetic disruption ofendogenous gene (e.g., TRAC, TRBC, PDCD1 or TIM3) was introduced containthe genetic disruption, do not express the targeted endogenouspolypeptide, or do not contain a contiguous and/or functional copy ofthe targeted gene. In some embodiments, the methods, compositions andcells according to the present disclosure include those in which atleast or greater than about 50%, 60%, 65%, 70%. 75%, 80%, 85%, 90% or95% of cells in a composition of cells into which an agent (e.g.gRNA/Cas9) for knockout or genetic disruption of a targeted gene wasintroduced do not express the targeted polypeptide, such as on thesurface of the immune cells. In some embodiments, at least or greaterthan about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of cells in acomposition of cells into which an agent (e.g. gRNA/Cas9) for knockoutor genetic disruption of the targeted gene was introduced are knockedout in both alleles, i.e. comprise a biallelic deletion, in suchpercentage of cells.

In some embodiments, provided are compositions and methods in which theCas9-mediated cleavage efficiency (% indel) in or near the targeted gene(e.g. within or about within 100 base pairs, within or about within 50base pairs, or within or about within 25 base pairs or within or aboutwithin 10 base pairs upstream or downstream of the cut site) is at leastor greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% incells of a composition of cells into which an agent (e.g. gRNA/Cas9) forknockout or genetic disruption of a targeted gene has been introduced.

In some embodiments, the provided cells, compositions and methodsresults in a reduction or disruption of signals delivered via theendogenous gene in at least or greater than about 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% or 95% of cells in a composition of cells into whichan agent (e.g. gRNA/Cas9) for knockout or genetic disruption of atargeted gene was introduced.

In some embodiments, compositions according to the provided disclosurethat comprise cells engineered with a recombinant receptor and comprisethe reduction, deletion, elimination, knockout or disruption inexpression of an endogenous receptor (e.g. genetic disruption of a TRBC,TRAC or immune checkpoint gene) retain the functional property oractivities of the receptor compared to the receptor expressed inengineered cells of a corresponding or reference composition comprisingthe receptor but do not comprise the genetic disruption of a gene orexpress the polypeptide when assessed under the same conditions. In someembodiments, the engineered cells of the provided compositions retain afunctional property or activity compared to a corresponding or referencecomposition comprising engineered cells in which such are engineeredwith the recombinant receptor but do not comprise the genetic disruptionor express the targeted polypeptide when assessed under the sameconditions. In some embodiments, the cells retain cytotoxicity,proliferation, survival or cytokine secretion compared to such acorresponding or reference composition.

In some embodiments, the immune cells in the composition retain aphenotype of the immune cell or cells compared to the phenotype of cellsin a corresponding or reference composition when assessed under the sameconditions. In some embodiments, cells in the composition include naivecells, effector memory cells, central memory cells, stem central memorycells, effector memory cells, and long-lived effector memory cells. Insome embodiments, the percentage of T cells, or T cells expressing therecombinant receptor (e.g. NY-ESO-1 TCR), and comprising the geneticdisruption of a targeted gene (e.g., TRAC, TRBC, PDCD1, or TIM3) exhibita non-activated, long-lived memory or central memory phenotype that isthe same or substantially the same as a corresponding or referencepopulation or composition of cells engineered with the recombinantreceptor but not containing the genetic disruption or expressing theswitch receptor. In some embodiments, such property, activity orphenotype can be measured in an in vitro assay, such as by incubation ofthe cells in the presence of the NY-ESO-1 antigen, a cell expressing theantigen and/or an antigen-receptor activating substance. In someembodiments, any of the assessed activities, properties or phenotypescan be assessed at various days following electroporation or otherintroduction of the agent, such as after or up to 3, 4, 5, 6, 7 days. Insome embodiments, such activity, property or phenotype is retained by atleast 80%, 85%, 90%, 95% or 100% of the cells in the compositioncompared to the activity of a corresponding composition containing cellsengineered with the recombinant receptor but not comprising the geneticdisruption of the targeted gene when assessed under the same conditions.

As used herein, reference to a “corresponding composition” or a“corresponding population of immune cells” (also called a “referencecomposition” or a “reference population of cells”) refers to immunecells (e.g., T cells) obtained, isolated, generated, produced and/orincubated under the same or substantially the same conditions, exceptthat the immune cells or population of immune cells were not introducedwith the agent. In some aspects, except for not containing introductionof the agent, such immune cells are treated identically or substantiallyidentically as immune cells that have been introduced with the agent,such that any one or more conditions that can influence the activity orproperties of the cell, including the upregulation or expression of theinhibitory molecule, is not varied or not substantially varied betweenthe cells other than the introduction of the agent.

Methods and techniques for assessing the expression and/or levels of Tcell markers are known in the art. Antibodies and reagents for detectionof such markers are well known in the art, and readily available. Assaysand methods for detecting such markers include, but are not limited to,flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT,cytometric bead array or other multiplex methods, Western Blot and otherimmunoaffinity-based methods. In some embodiments, antigen receptor(e.g. NY-ESO1 TCR)-expressing cells can be detected by flow cytometry orother immunoaffinity based method for expression of a marker unique tosuch cells, and then such cells can be co-stained for another T cellsurface marker or markers.

In some embodiments, the cells, compositions and methods provide for thedeletion, knockout, disruption, or reduction in expression of the targetgene in immune cells (e.g. T cells) to be adoptively transferred (suchas cells engineered to express exogenous NY-ESO-1 TCR). In someembodiments, the methods are performed ex vivo on primary cells, such asprimary immune cells (e.g. T cells) from a subject. In some aspects,methods of producing or generating such genetically engineered T cellsinclude introducing into a population of cells containing immune cells(e.g. T cells) one or more nucleic acids encoding a recombinant receptor(e.g. exogenous NY-ESO-1 TCR) and an agent or agents that is capable ofdisrupting, a gene that encode the endogenous receptor to be targeted.As used herein, the term “introducing” encompasses a variety of methodsof introducing DNA into a cell, either in vitro or in vivo, such methodsincluding transformation, transduction, transfection (e.g.electroporation), and infection. Vectors are useful for introducing DNAencoding molecules into cells. Possible vectors include plasmid vectorsand viral vectors. Viral vectors include retroviral vectors, lentiviralvectors, or other vectors such as adenoviral vectors or adeno-associatedvectors.

The population of cells containing T cells can be cells that have beenobtained from a subject, such as obtained from a peripheral bloodmononuclear cells (PBMC) sample, an unfractionated T cell sample, alymphocyte sample, a white blood cell sample, an apheresis product, or aleukapheresis product. In some embodiments, T cells can be separated orselected to enrich T cells in the population using positive or negativeselection and enrichment methods. In some embodiments, the populationcontains CD4+, CD8+ or CD4+ and CD8+ T cells. In some embodiments, thestep of introducing the nucleic acid encoding a genetically engineeredantigen receptor and the step of introducing the agent (e.g. Cas9/gRNARNP) can occur simultaneously or sequentially in any order. In someembodiments, subsequent to introduction of the exogenous receptor andone or more gene editing agents (e.g. Cas9/gRNA RNP), the cells arecultured or incubated under conditions to stimulate expansion and/orproliferation of cells.

Thus, provided are cells, compositions and methods that enhance immunecell, such as T cell, function in adoptive cell therapy, including thoseoffering improved efficacy, such as by increasing activity and potencyof administered genetically engineered (e.g. anti-NY-ESO-1) cells, whilemaintaining persistence or exposure to the transferred cells over time.In some embodiments, the genetically engineered cells, exhibit increasedexpansion and/or persistence when administered in vivo to a subject, ascompared to certain available methods. In some embodiments, the providedimmune cells exhibit increased persistence when administered in vivo toa subject. In some embodiments, the persistence of geneticallyengineered immune cells, in the subject upon administration is greateras compared to that which would be achieved by alternative methods, suchas those involving administration of cells genetically engineered bymethods in which T cells were not introduced with an agent that reducesexpression of or disrupts a gene encoding an endogenous receptor. Insome embodiments, the persistence is increased at least or about atleast 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, 100-fold or more.

In some embodiments, the degree or extent of persistence of administeredcells can be detected or quantified after administration to a subject.For example, in some aspects, quantitative PCR (qPCR) is used to assessthe quantity of cells expressing the exogenous receptor (e.g., NY-ESO-1TCR) in the blood or serum or organ or tissue (e.g., disease site) ofthe subject. In some aspects, persistence is quantified as copies of DNAor plasmid encoding the exogenous receptor per microgram of DNA, or asthe number of receptor-expressing cells per microliter of the sample,e.g., of blood or serum, or per total number of peripheral bloodmononuclear cells (PBMCs) or white blood cells or T cells per microliterof the sample. In some embodiments, flow cytometric assays detectingcells expressing the receptor generally using antibodies specific forthe receptors also can be performed. Cell-based assays may also be usedto detect the number or percentage of functional cells, such as cellscapable of binding to and/or neutralizing and/or inducing responses,e.g., cytotoxic responses, against cells of the disease or condition orexpressing the antigen recognized by the receptor. In any of suchembodiments, the extent or level of expression of another markerassociated with the exogenous receptor (e.g. exogenous NY-ESO-1 TCR) canbe used to distinguish the administered cells from endogenous cells in asubject.

F. Methods of Producing Genetically Modified Immune Cells

The present disclosure provides methods for producing or generating amodified immune cell or precursor thereof (e.g., a T cell) of theinvention for tumor immunotherapy, e.g., adoptive immunotherapy. Thecells generally are engineered by introducing one or more geneticallyengineered nucleic acids encoding the exogenous receptors (e.g., aNY-ESO-1 receptor and/or a switch receptor). In some embodiments, thecells also are introduced, either simultaneously or sequentially withthe nucleic acid encoding the exogenous receptor, with an agent (e.g.Cas9/gRNA RNP) that is capable of disrupting a targeted gene (e.g., agene encoding TRAC, TRBC or an immune inhibitory molecule such as PD-1.

In some embodiments, the exogenous receptor (e.g., TCR and/or switchreceptor) is introduced into a cell by an expression vector. Expressionvectors comprising a nucleic acid sequence encoding a TCR and/or switchreceptor of the present invention are provided herein. Suitableexpression vectors include lentivirus vectors, gamma retrovirus vectors,foamy virus vectors, adeno associated virus (AAV) vectors, adenovirusvectors, engineered hybrid viruses, naked DNA, including but not limitedto transposon mediated vectors, such as Sleeping Beauty, Piggybak, andIntegrases such as Phi31. Some other suitable expression vectors includeHerpes simplex virus (HSV) and retrovirus expression vectors.

Adenovirus expression vectors are based on adenoviruses, which have alow capacity for integration into genomic DNA but a high efficiency fortransfecting host cells. Adenovirus expression vectors containadenovirus sequences sufficient to: (a) support packaging of theexpression vector and (b) to ultimately express the TCR and/or switchreceptor in the host cell. In some embodiments, the adenovirus genome isa 36 kb, linear, double stranded DNA, where a foreign DNA sequence(e.g., a nucleic acid encoding an exogenous TCR and/or switch receptor)may be inserted to substitute large pieces of adenoviral DNA in order tomake the expression vector of the present invention (see, e.g.,Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

Another expression vector is based on an adeno associated virus, whichtakes advantage of the adenovirus coupled systems. This AAV expressionvector has a high frequency of integration into the host genome. It caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue cultures or in vivo. TheAAV vector has a broad host range for infectivity. Details concerningthe generation and use of AAV vectors are described in U.S. Pat. Nos.5,139,941 and 4,797,368.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retrovirus vector is constructed by inserting a nucleicacid (e.g., a nucleic acid encoding an exogenous TCR and/or switchreceptor) into the viral genome at certain locations to produce a virusthat is replication defective. Though the retrovirus vectors are able toinfect a broad variety of cell types, integration and stable expressionof the TCR and/or switch receptor requires the division of host cells.

Lentivirus vectors are derived from lentiviruses, which are complexretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2)and the Simian Immunodeficiency Virus (SIV). Lentivirus vectors havebeen generated by multiply attenuating the HIV virulence genes, forexample, the genes env, vif, vpr, vpu and nef are deleted making thevector biologically safe. Lentivirus vectors are capable of infectingnon-dividing cells and can be used for both in vivo and ex vivo genetransfer and expression, e.g., of a nucleic acid encoding a TCR and/or aswitch receptor (see, e.g., U.S. Pat. No. 5,994,136).

Expression vectors including a nucleic acid of the present disclosurecan be introduced into a host cell by any means known to persons skilledin the art. The expression vectors may include viral sequences fortransfection, if desired. Alternatively, the expression vectors may beintroduced by fusion, electroporation, biolistics, transfection,lipofection, or the like. The host cell may be grown and expanded inculture before introduction of the expression vectors, followed by theappropriate treatment for introduction and integration of the vectors.The host cells are then expanded and may be screened by virtue of amarker present in the vectors. Various markers that may be used areknown in the art, and may include hprt, neomycin resistance, thymidinekinase, hygromycin resistance, etc. As used herein, the terms “cell,”“cell line,” and “cell culture” may be used interchangeably. In someembodiments, the host cell is an immune cell or precursor thereof, e.g.,a T cell, an NK cell, or an NKT cell.

The present invention also provides genetically engineered cells whichinclude and stably express a TCR and/or switch receptor of the presentdisclosure. In some embodiments, the genetically engineered cells aregenetically engineered T-lymphocytes (T cells), naive T cells (TN),memory T cells (for example, central memory T cells (TCM), effectormemory cells (TEM)), natural killer cells (NK cells), and macrophagescapable of giving rise to therapeutically relevant progeny. In oneembodiment, the genetically engineered cells are autologous cells.

Modified cells (e.g., comprising a TCR and/or a switch receptor) may beproduced by stably transfecting host cells with an expression vectorincluding a nucleic acid of the present disclosure. Additional methodsto generate a modified cell of the present disclosure include, withoutlimitation, chemical transformation methods (e.g., using calciumphosphate, dendrimers, liposomes and/or cationic polymers), non-chemicaltransformation methods (e.g., electroporation, optical transformation,gene electrotransfer and/or hydrodynamic delivery) and/or particle-basedmethods (e.g., impalefection, using a gene gun and/or magnetofection).Transfected cells expressing a TCR and/or switch receptor of the presentdisclosure may be expanded ex vivo.

Physical methods for introducing an expression vector into host cellsinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells including vectors and/or exogenous nucleic acids arewell-known in the art. See, e.g., Sambrook et al. (2001), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.Chemical methods for introducing an expression vector into a host cellinclude colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K &K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,AL). Stock solutions of lipids in chloroform or chloroform/methanol canbe stored at about −20° C. Chloroform may be used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). Compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, molecular biology assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;biochemistry assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, the nucleic acids introduced into the host cell areRNA. In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA may be produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA may be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA.

PCR may be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary,” as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary. Substantiallycomplementary sequences are able to anneal or hybridize with theintended DNA target under annealing conditions used for PCR. The primerscan be designed to be substantially complementary to any portion of theDNA template. For example, the primers can be designed to amplify theportion of a gene that is normally transcribed in cells (the openreading frame), including 5 and 3′ UTRs. The primers may also bedesigned to amplify a portion of a gene that encodes a particular domainof interest. In one embodiment, the primers are designed to amplify thecoding region of a human cDNA, including all or portions of the 5′ and3′ UTRs. Primers useful for PCR are generated by synthetic methods thatare well known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5 and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5 end and a 3′ poly(A)tail which determine ribosome binding, initiation of translation andstability mRNA in the cell. On a circular DNA template, for instance,plasmid DNA, RNA polymerase produces a long concatameric product whichis not suitable for expression in eukaryotic cells. The transcription ofplasmid DNA linearized at the end of the 3′ UTR results in normal sizedmRNA which is not effective in eukaryotic transfection even if it ispolyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA. 5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

In some embodiments, the RNA is electroporated into the cells, such asin vitro transcribed RNA. Any solutes suitable for cell electroporation,which can contain factors facilitating cellular permeability andviability such as sugars, peptides, lipids, proteins, antioxidants, andsurfactants can be included.

In some embodiments, a nucleic acid encoding a TCR and/or a switchreceptor of the present disclosure will be RNA, e.g., in vitrosynthesized RNA. Methods for in vitro synthesis of RNA are known in theart; any known method can be used to synthesize RNA comprising asequence encoding a TCR and/or a switch receptor. Methods forintroducing RNA into a host cell are known in the art. See, e.g., Zhaoet al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising anucleotide sequence encoding a TCR and/or a switch receptor into a hostcell can be carried out in vitro or ex vivo or in vivo. For example, ahost cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can beelectroporated in vitro or ex vivo with RNA comprising a nucleotidesequence encoding a TCR and/or a switch receptor.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. A RNA transgenecan be delivered to a lymphocyte and expressed therein following a briefin vitro cell activation, as a minimal expressing cassette without theneed for any additional viral sequences. Under these conditions,integration of the transgene into the host cell genome is unlikely.Cloning of cells is not necessary because of the efficiency oftransfection of the RNA and its ability to uniformly modify the entirelymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

In another aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

In some embodiments, the immune cells (e.g. T cells) can be incubated orcultivated prior to, during and/or subsequent to introducing the nucleicacid molecule encoding the exogenous receptor (e.g., the exogenousNY-ESO-1 receptor and/or switch receptor) and the gene editing agent(e.g. CRISPR system, CRISPR/Cas9 system, Cas9/gRNA RNP). In someembodiments, the cells (e.g. T cells) can be incubated or cultivatedprior to, during or subsequent to the introduction of the nucleic acidmolecule encoding the exogenous receptor, such as prior to, during orsubsequent to the transduction of the cells with a viral vector (e.g.lentiviral vector) encoding the exogenous receptor. In some embodiments,the cells (e.g. T cells) can be incubated or cultivated prior to, duringor subsequent to the introduction of the gene editing agent (e.g.Cas9/gRNA RNP), such as prior to, during or subsequent to contacting thecells with the agent or prior to, during or subsequent to delivering theagent into the cells, e.g. via electroporation. In some embodiments, theincubation can be both in the context of introducing the nucleic acidmolecule encoding the exogenous receptor and introducing the geneediting agent, e.g. Cas9/gRNA RNP. In some embodiments, the methodincludes activating or stimulating cells with a stimulating oractivating agent (e.g. anti-CD3/anti-CD28 antibodies) prior tointroducing the nucleic acid molecule encoding the exogenous receptorand the gene editing agent, e.g. Cas9/gRNA RNP.

In some embodiments, the introducing the gene editing agent, e.g.Cas9/gRNA RNP, is after introducing the nucleic acid molecule encodingthe exogenous receptor. In some embodiments, prior to the introducing ofthe agent, the cells are rested, e.g. by removal of any stimulating oractivating agent. In some embodiments, prior to introducing the agent,the stimulating or activating agent and/or cytokines are not removed.

G. Sources of Immune Cells

Prior to expansion, a source of immune cells is obtained from a subjectfor ex vivo manipulation. Sources of target cells for ex vivomanipulation may also include, e.g., autologous or heterologous donorblood, cord blood, or bone marrow. For example the source of immunecells may be from the subject to be treated with the modified immunecells of the invention, e.g., the subject's blood, the subject's cordblood, or the subject's bone marrow. Non-limiting examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood,peripheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cellsare cells of the immune system, such as cells of the innate or adaptiveimmunity, e.g., myeloid or lymphoid cells, including lymphocytes,typically T cells and/or NK cells. Other exemplary cells include stemcells, such as multipotent and pluripotent stem cells, including inducedpluripotent stem cells (iPSCs). In some aspects, the cells are humancells. With reference to the subject to be treated, the cells may beallogeneic and/or autologous. The cells typically are primary cells,such as those isolated directly from a subject and/or isolated from asubject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell(e.g., a CD8+ naive T cell, central memory T cell, or effector memory Tcell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatoryT cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell ahematopoietic stem cell, a natural killer cell (NK cell) or a dendriticcell. In some embodiments, the cells are monocytes or granulocytes,e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, and/or basophils. In an embodiment, the target cellis an induced pluripotent stem (iPS) cell or a cell derived from an iPScell, e.g., an iPS cell generated from a subject, manipulated to alter(e.g., induce a mutation in) or manipulate the expression of one or moretarget genes, and differentiated into, e.g., a T cell, e.g., a CD8+ Tcell (e.g., a CD8+ naive T cell, central memory T cell, or effectormemory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoidprogenitor cell or a hematopoietic stem cell.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. Among the sub-types and subpopulations of Tcells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells,effector T cells (TEFF), memory T cells and sub-types thereof, such asstem cell memory T (TSCM), central memory T (TCM), effector memory T(TEM), or terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT)cells, naturally occurring and adaptive regulatory T (Treg) cells,helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9cells, TH22 cells, follicular helper T cells, alpha/beta T cells, anddelta/gamma T cells. In certain embodiments, any number of T cell linesavailable in the art, may be used.

In some embodiments, the methods include isolating immune cells from thesubject, preparing, processing, culturing, and/or engineering them. Insome embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for engineering asdescribed may be isolated from a sample, such as a biological sample,e.g., one obtained from or derived from a subject. In some embodiments,the subject from which the cell is isolated is one having the disease orcondition or in need of a cell therapy or to which cell therapy will beadministered. The subject in some embodiments is a human in need of aparticular therapeutic intervention, such as the adoptive cell therapyfor which cells are being isolated, processed, and/or engineered.Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig. Insome embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets. In some embodiments, the blood cellscollected from the subject are washed, e.g., to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In some embodiments, the cells are washedwith phosphate buffered saline (PBS). In some aspects, a washing step isaccomplished by tangential flow filtration (TFF) according to themanufacturer's instructions. In some embodiments, the cells areresuspended in a variety of biocompatible buffers after washing. Incertain embodiments, components of a blood cell sample are removed andthe cells directly resuspended in culture media. In some embodiments,the methods include density-based cell separation methods, such as thepreparation of white blood cells from peripheral blood by lysing the redblood cells and centrifugation through a Percoll or Ficoll gradient.

In one embodiment, immune are obtained cells from the circulating bloodof an individual by apheresis or leukapheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. The cells collected by apheresis may be washed toremove the plasma fraction and to place the cells in an appropriatebuffer or media, such as phosphate buffered saline (PBS) or washsolution that lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population. The separation need not result in 100%enrichment or removal of a particular cell population or cellsexpressing a particular marker. For example, positive selection of orenrichment for cells of a particular type, such as those expressing amarker, refers to increasing the number or percentage of such cells, butneed not result in a complete absence of cells not expressing themarker. Likewise, negative selection, removal, or depletion of cells ofa particular type, such as those expressing a marker, refers todecreasing the number or percentage of such cells, but need not resultin a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

In some embodiments, one or more of the T cell populations is enrichedfor or depleted of cells that are positive for (marker+) or express highlevels (marker^(high)) of one or more particular markers, such assurface markers, or that are negative for (marker −) or expressrelatively low levels (marker^(low)) of one or more markers. Forexample, in some aspects, specific subpopulations of T cells, such ascells positive or expressing high levels of one or more surface markers,e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/orCD45RO+ T cells, are isolated by positive or negative selectiontechniques. In some cases, such markers are those that are absent orexpressed at relatively low levels on certain populations of T cells(such as non-memory cells) but are present or expressed at relativelyhigher levels on certain other populations of T cells (such as memorycells). In one embodiment, the cells (such as the CD8+ cells or the Tcells, e.g., CD3+ cells) are enriched for (i.e., positively selectedfor) cells that are positive or expressing high surface levels ofCD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of(e.g., negatively selected for) cells that are positive for or expresshigh surface levels of CD45RA. In some embodiments, cells are enrichedfor or depleted of cells positive or expressing high surface levels ofCD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+T cells are enriched for cells positive for CD45RO (or negative forCD45RA) and for CD62L. For example, CD3+, CD28+ T cells can bepositively selected using CD3/CD28 conjugated magnetic beads (e.g.,DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4+ or CD8+ election step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations. In someembodiments, CD8+ cells are further enriched for or depleted of naive,central memory, effector memory, and/or central memory stem cells, suchas by positive or negative selection based on surface antigensassociated with the respective subpopulation. In some embodiments,enrichment for central memory T (TCM) cells is carried out to increaseefficacy, such as to improve long-term survival, expansion, and/orengraftment following administration, which in some aspects isparticularly robust in such sub-populations. In some embodiments,combining TCM-enriched CD8+ T cells and CD4+ T cells further enhancesefficacy.

In some embodiments, memory T cells are present in both CD62L+ andCD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can beenriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, suchas using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+T cell population and a CD8+ T cell sub-population, e.g., asub-population enriched for central memory (TCM) cells. In someembodiments, the enrichment for central memory T (TCM) cells is based onpositive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3,and/or CD127; in some aspects, it is based on negative selection forcells expressing or highly expressing CD45RA and/or granzyme B. In someaspects, isolation of a CD8+ population enriched for TCM cells iscarried out by depletion of cells expressing CD4, CD14, CD45RA, andpositive selection or enrichment for cells expressing CD62L. In oneaspect, enrichment for central memory T (TCM) cells is carried outstarting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ cell population or subpopulation, also is used to generate theCD4+ cell population or sub-population, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

CD4+ T helper cells are sorted into naive, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+ cells are CD62L+ andCD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO.In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CDI Ib, CD16, HLA-DR, and CD8. In some embodiments, the antibodyor binding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor. The conditions caninclude one or more of particular media, temperature, oxygen content,carbon dioxide content, time, agents, e.g., nutrients, amino acids,antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors, and any other agents designed to activate the cells.In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from an umbilical cord. In any event, a specificsubpopulation of T cells can be further isolated by positive or negativeselection techniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19, and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4⁺ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° C. per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

In one embodiment, the population of T cells is comprised within cellssuch as peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of T cells.In yet another embodiment, purified T cells comprise the population of Tcells.

In certain embodiments, T regulatory cells (Tregs) can be isolated froma sample. The sample can include, but is not limited to, umbilical cordblood or peripheral blood. In certain embodiments, the Tregs areisolated by flow-cytometry sorting. The sample can be enriched for Tregsprior to isolation by any means known in the art. The isolated Tregs canbe cryopreserved, and/or expanded prior to use. Methods for isolatingTregs are described in U.S. Pat. Nos. 7,754,482, 8,722,400, and9,555,105, and U.S. patent application Ser. No. 13/639,927, contents ofwhich are incorporated herein in their entirety.

H. Expansion of Immune Cells

Whether prior to or after modification of cells to express a TCR and/ora switch receptor, the cells can be activated and expanded in numberusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005.For example, the T cells of the invention may be expanded by contactwith a surface having attached thereto an agent that stimulates aCD3/TCR complex associated signal and a ligand that stimulates aco-stimulatory molecule on the surface of the T cells. In particular, Tcell populations may be stimulated by contact with an anti-CD3 antibody,or antigen-binding fragment thereof, or an anti-CD2 antibody immobilizedon a surface, or by contact with a protein kinase C activator (e.g.,bryostatin) in conjunction with a calcium ionophore. For co-stimulationof an accessory molecule on the surface of the T cells, a ligand thatbinds the accessory molecule is used. For example, T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,Besancon, France) and these can be used in the invention, as can othermethods and reagents known in the art (see, e.g., ten Berge et al.,Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med.(1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999)227(1-2): 53-63).

Expanding T cells by the methods disclosed herein can be multiplied byabout 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold,4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and anyand all whole or partial integers therebetween. In one embodiment, the Tcells expand in the range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the TCR and/or switch receptor.

Another procedure for ex vivo expansion of cells is described in U.S.Pat. No. 5,199,942 (incorporated herein by reference). Expansion, suchas described in U.S. Pat. No. 5,199,942 can be an alternative or inaddition to other methods of expansion described herein. Briefly, exvivo culture and expansion of T cells comprises the addition to thecellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kitligand. In one embodiment, expanding the T cells comprises culturing theT cells with a factor selected from the group consisting of flt3-L,IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. A cell isolated by the methods disclosed herein can beexpanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold,500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold,3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, orgreater. In one embodiment, the T cells expand in the range of about 20fold to about 50 fold, or more. In one embodiment, human T regulatorycells are expanded via anti-CD3 antibody coated KT64.86 artificialantigen presenting cells (aAPCs). Methods for expanding and activating Tcells can be found in U.S. Pat. Nos. 7,754,482, 8,722,400, and9,555,105, contents of which are incorporated herein in their entirety.

In one embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. Inanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as a transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid, into the expanded population of Tcells, wherein the agent further stimulates the T cell. The agent maystimulate the T cells, such as by stimulating further expansion,effector function, or another T cell function.

I. Methods of Treatment

The modified cells (e.g., T cells) described herein may be included in acomposition for immunotherapy. The composition may include apharmaceutical composition and further include a pharmaceuticallyacceptable carrier. A therapeutically effective amount of thepharmaceutical composition comprising the modified T cells may beadministered.

In one aspect, the invention includes a method for adoptive celltransfer therapy comprising administering to a subject in need thereof amodified T cell of the present invention. In another aspect, theinvention includes a method of treating a disease or condition in asubject comprising administering to a subject in need thereof apopulation of modified T cells.

Also included is a method of treating a disease or condition in asubject in need thereof comprising administering to the subject agenetically edited modified cell (e.g., genetically edited modified Tcell). In one embodiment, the method of treating a disease or conditionin a subject in need thereof comprises administering to the subject agenetically edited modified cell comprising an exogenous TCR (e.g., agenetically modified T cell comprising an exogenous TCR having affinityfor NY-ESO-1 on a target cell). In one embodiment, the method oftreating a disease or condition in a subject in need thereof comprisesadministering to the subject a genetically editing modified cellcomprising an exogenous TCR and a switch receptor (e.g., a geneticallymodified T cell comprising an exogenous TCR having affinity for NY-ESO-1on a target cell and a PD1-CD28 switch receptor).

Also provided are methods of treating cancer in a subject in needthereof comprising administering to the subject a composition comprisingany of the modified T cells of the present invention.

In one aspect, the invention includes a method of treating multiplemyeloma in a subject in need thereof. The method comprises administeringto the subject a lymphodepleting chemotherapy comprising an effectiveamount of cyclophosphamide and a modified T cell. The modified T cellcomprises an exogenous T cell receptor (TCR) having affinity forNY-ESO-1 on a target cell. The exogenous TCR comprises a TCR alpha chaincomprising the amino acid sequence set forth in SEQ ID NO:5 and a TCRbeta chain comprising the amino acid sequence set forth in SEQ ID NO:12.The T cell also comprises at least one nucleotide substitution,deletion, insertion, and/or insertion/deletion in an endogenous TCRalpha chain coding sequence comprising the nucleic acid sequence setforth in SEQ ID NO:128, in an endogenous TCR beta chain coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:129, and inan endogenous PD1 coding sequence comprising the nucleic acid sequenceset forth in SEQ ID NO:130. The expression of the endogenous TCR alphachain coding sequence, the endogenous TCR beta chain coding sequence,and endogenous PD1 coding sequence are downregulated.

In another aspect, the invention includes a method of treating melanoma,synovial sarcoma, or myxoid/round cell liposarcoma in a subject in needthereof. The method comprises administering to the subject alymphodepleting chemotherapy, which comprises an effective amount ofcyclophosphamide and an effective amount of fludarabine, and a modifiedT cell. The modified T cell comprises an exogenous T cell receptor (TCR)having affinity for NY-ESO-1 on a target cell. The exogenous TCRcomprises a TCR alpha chain comprising the amino acid sequence set forthin SEQ ID NO:5 and a TCR beta chain comprising the amino acid sequenceset forth in SEQ ID NO:12. The T cell also comprises at least onenucleotide substitution, deletion, insertion, and/or insertion/deletionin an endogenous TCR alpha chain coding sequence comprising the nucleicacid sequence set forth in SEQ ID NO:128, in an endogenous TCR betachain coding sequence comprising the nucleic acid sequence set forth inSEQ ID NO:129, and in an endogenous PD1 coding sequence comprising thenucleic acid sequence set forth in SEQ ID NO:130. The expression of theendogenous TCR alpha chain coding sequence, the endogenous TCR betachain coding sequence, and endogenous PD1 coding sequence aredownregulated.

Methods for administration of immune cells for adoptive cell therapy areknown and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, the cell therapy, e.g., adoptive T celltherapy is carried out by autologous transfer, in which the cells areisolated and/or otherwise prepared from the subject who is to receivethe cell therapy, or from a sample derived from such a subject. Thus, insome aspects, the cells are derived from a subject, e.g., patient, inneed of a treatment and the cells, following isolation and processingare administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In some embodiments, the subject has been treated with a therapeuticagent targeting the disease or condition, e.g. the tumor, prior toadministration of the cells or composition containing the cells. In someaspects, the subject is refractory or non-responsive to the othertherapeutic agent. In some embodiments, the subject has persistent orrelapsed disease, e.g., following treatment with another therapeuticintervention, including chemotherapy, radiation, and/or hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT. In someembodiments, the administration effectively treats the subject despitethe subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some aspects, the subject is initially responsive to the therapeuticagent, but exhibits a relapse of the disease or condition over time. Insome embodiments, the subject has not relapsed. In some suchembodiments, the subject is determined to be at risk for relapse, suchas at a high risk of relapse, and thus the cells are administeredprophylactically, e.g., to reduce the likelihood of or prevent relapse.In some aspects, the subject has not received prior treatment withanother therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapeutic intervention,including chemotherapy, radiation, and/or hematopoietic stem celltransplantation (HSCT), e.g., allogenic HSCT. In some embodiments, theadministration effectively treats the subject despite the subject havingbecome resistant to another therapy.

The modified immune cells of the present invention can be administeredto an animal, preferably a mammal, even more preferably a human, totreat a cancer. In addition, the cells of the present invention can beused for the treatment of any condition related to a cancer, especiallya cell-mediated immune response against a tumor cell(s), where it isdesirable to treat or alleviate the disease. The types of cancers to betreated with the modified cells or pharmaceutical compositions of theinvention include, carcinoma, blastoma, and sarcoma, and certainleukemia or lymphoid malignancies, benign and malignant tumors, andmalignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplarycancers include but are not limited to breast cancer, prostate cancer,ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, thyroid cancer, and the like. The cancers may benon-solid tumors (such as hematological tumors) or solid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included. In oneembodiment, the cancer is a solid tumor or a hematological tumor. In oneembodiment, the cancer is a carcinoma. In one embodiment, the cancer isa sarcoma. In one embodiment, the cancer is a leukemia. In oneembodiment the cancer is a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a myeloma, or a condition related tomyeloma. Examples of myeloma or conditions related thereto include,without limitation, light chain myeloma, non-secretory myeloma,monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma(e.g., solitary, multiple solitary, extramedullary plasmacytoma),amyloidosis, and multiple myeloma. In one embodiment, a method of thepresent disclosure is used to treat multiple myeloma. In one embodiment,a method of the present disclosure is used to treat refractory myeloma.In one embodiment, a method of the present disclosure is used to treatrelapsed myeloma.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a melanoma, or a condition related tomelanoma. Examples of melanoma or conditions related thereto include,without limitation, superficial spreading melanoma, nodular melanoma,lentigo maligna melanoma, acral lentiginous melanoma, amelanoticmelanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina,rectum melanoma). In one embodiment, a method of the present disclosureis used to treat cutaneous melanoma. In one embodiment, a method of thepresent disclosure is used to treat refractory melanoma. In oneembodiment, a method of the present disclosure is used to treat relapsedmelanoma.

In yet other exemplary embodiments, the modified immune cells of theinvention are used to treat a sarcoma, or a condition related tosarcoma. Examples of sarcoma or conditions related thereto include,without limitation, angiosarcoma, chondrosarcoma, chordoma, Ewing'ssarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, fibrosarcoma, gastrointestinal stromaltumor, leiomyosarcoma, liposarcoma, mesothelioma, malignant peripheralnerve sheath tumor, myxosarcoma, osteogenic sarcoma, osteosarcoma,pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, synovioma, andother soft tissue sarcomas. In one embodiment, a method of the presentdisclosure is used to treat synovial sarcoma. In one embodiment, amethod of the present disclosure is used to treat liposarcoma such asmyxoid/round cell liposarcoma, differentiated/dedifferentiatedliposarcoma, and pleomorphic liposarcoma. In one embodiment, a method ofthe present disclosure is used to treat myxoid/round cell liposarcoma.In one embodiment, a method of the present disclosure is used to treat arefractory sarcoma. In one embodiment, a method of the presentdisclosure is used to treat a relapsed sarcoma.

In certain embodiments, the subject is provided a secondary treatment.Secondary treatments include but are not limited to chemotherapy,radiation, surgery, and medications.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of thecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

The cells of the invention to be administered may be autologous, withrespect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out inany convenient manner known to those of skill in the art. The cells ofthe present invention may be administered to a subject by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4+ to CD8+ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8+and CD4+ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsubtype, or minimum number of cells of the population or sub-type perunit of body weight. Thus, in some embodiments, the dosage is based on adesired fixed dose of total cells and a desired ratio, and/or based on adesired fixed dose of one or more, e.g., each, of the individualsub-types or sub-populations. Thus, in some embodiments, the dosage isbased on a desired fixed or minimum dose of T cells and a desired ratioof CD4⁺ to CD8⁺ cells, and/or is based on a desired fixed or minimumdose of CD4⁺ and/or CD8⁺ cells.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells, such as, e.g., 1 million toabout 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), such asabout 10 million to about 100 billion cells (e.g., about 20 millioncells, about 30 million cells, about 40 million cells, about 60 millioncells, about 70 million cells, about 80 million cells, about 90 millioncells, about 10 billion cells, about 25 billion cells, about 50 billioncells, about 75 billion cells, about 90 billion cells, or a rangedefined by any two of the foregoing values), and in some cases about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about 1×10⁵cells/kg to about 1×10¹¹ cells/kg 10⁴ and at or about 10¹¹cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ cells/kgbody weight, for example, at or about 1×10⁵ cells/kg, 1.5×10⁵ cells/kg,2×10⁵ cells/kg, or 1×10⁶ cells/kg body weight. For example, in someembodiments, the cells are administered at, or within a certain range oferror of, between at or about 10⁴ and at or about 10⁹ T cells/kilograms(kg) body weight, such as between 10⁵ and 10⁶ T cells/kg body weight,for example, at or about 1×10⁵ T cells/kg, 1.5×10⁵ T cells/kg, 2×10⁵ Tcells/kg, or 1×10⁶ T cells/kg body weight. In other exemplaryembodiments, a suitable dosage range of modified cells for use in amethod of the present disclosure includes, without limitation, fromabout 1×10⁵ cells/kg to about 1×10⁶ cells/kg, from about 1×10⁶ cells/kgto about 1×10⁷ cells/kg, from about 1×10⁷ cells/kg about 1×10⁸ cells/kg,from about 1×10⁸ cells/kg about 1×10⁹ cells/kg, from about 1×10⁹cells/kg about 1×10¹⁰ cells/kg, from about 1×10¹⁰ cells/kg about 1×10¹¹cells/kg. In an exemplary embodiment, a suitable dosage for use in amethod of the present disclosure is about 1×10⁸ cells/kg. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 1×10⁷ cells/kg. In other embodiments, asuitable dosage is from about 1×10⁷ total cells to about 5×10⁷ totalcells. In some embodiments, a suitable dosage is from about 1×10⁸ totalcells to about 5×10⁸ total cells. In some embodiments, a suitable dosageis from about 1.4×10⁷ total cells to about 1.1×10⁹ total cells. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 7×10⁹ total cells.

In some embodiments, the cells are administered at or within a certainrange of error of between at or about 10⁴ and at or about 10⁹ CD4⁺and/or CD8⁺ cells/kilograms (kg) body weight, such as between 10⁵ and10⁶ CD4⁺ and/or CD8⁺ cells/kg body weight, for example, at or about1×10⁵ CD4⁺ and/or CD8⁺ cells/kg, 1.5×10⁵ CD4⁺ and/or CD8⁺ cells/kg,2×10⁵ CD4⁺ and/or CD8⁺ cells/kg, or 1×10⁶ CD4⁺ and/or CD8⁺ cells/kg bodyweight. In some embodiments, the cells are administered at or within acertain range of error of, greater than, and/or at least about 1×10⁶,about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ CD4⁺ cells,and/or at least about 1×10⁶, about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶,or about 9×10⁶ CD8⁺ cells, and/or at least about 1×10⁶, about 2.5×10⁶,about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ T cells. In some embodiments,the cells are administered at or within a certain range of error ofbetween about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ t cells,between about 10⁹ and 10¹² or between about 10¹⁰ and 10¹¹ CD4⁺ cells,and/or between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD8⁺cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios,for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

In some embodiments, a dose of modified cells is administered to asubject in need thereof, in a single dose or multiple doses. In someembodiments, a dose of modified cells is administered in multiple doses,e.g., once a week or every 7 days, once every 2 weeks or every 14 days,once every 3 weeks or every 21 days, once every 4 weeks or every 28days. In an exemplary embodiment, a single dose of modified cells isadministered to a subject in need thereof. In an exemplary embodiment, asingle dose of modified cells is administered to a subject in needthereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence. In some embodiments,the methods comprise administration of a chemotherapeutic agent.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable method knownin the art, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al. J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load.

In some exemplary embodiments, the methods of the invention employspecific dosage regimen to treat a subject in need thereof. In anexemplary embodiment, a suitable subject is an adult HLA-A*0201 positivepatient with relapsed or refractory tumors expressing NY-ESO-1 antigen.In one embodiment, to identify such patients, a basket design enrollingpatients with myeloma, synovial sarcoma, and myxoid/round cellliposarcoma (MRCL), and melanoma may be used. In one embodiment patientswith multiple myeloma and sarcoma may be targeted for enrollment first,with the plan to expand enrollment to include melanoma patients onceinitial safety is established.

In some exemplary embodiments, a suitable subject is an HLA-A*0201positive patient with tumors expressing NY-ESO-1 antigen. As describedherein, the NY-ESO-1 derived peptide in complex with HLA-A*0201 isSLLMWITQC (NY-ESO₁₅₇₋₁₆₅; SEQ ID NO: 1). The Cancer Testis AntigenLAGE-1 is 90% homologous to NY-ESO-1, and has two transcript variants,LAGE-1S and LAGE-1L. The LAGE-1S transcript variant shares the sameHLA-A*0201 epitope, SLLMWITQC (SEQ ID NO: 1), with NY-ESO-1. In someexemplary embodiments, the LAGE-1 HLA-A*0201 epitope is an equivalenttarget for NY-ESO-1 directed therapy. Accordingly, in some exemplaryembodiments, a suitable subject for NY-ESO-1 directed therapy is anHLA-A*0201 positive patient with tumors expressing LAGE-1 antigen. See,e.g., Rapoport et al., Nature Medicine (2015) 21(8): 914-921; Rimoldi etal., J. Immunology (2000) 165(12): 7253-7261; and Purbhoo et al., J.Immunology (2006) 176(12): 7308-7316.

In some exemplary embodiments, identification of a subject suitable fortreatment comprises screening for HLA-A*0201 expression, and expressionof NY-ESO-1 and/or LAGE-1. Screening methods are known in the art. Forexample, PCR, or RT-PCR can be performed to identify patients that arepositive for expression of NY-ESO-1 and/or LAGE-1. In some cancers,e.g., multiple myeloma, both NY-ESO-1 and LAGE-1 are expressed by thecancer cells, and LAGE-1 is commonly found to be expressed at a higherlevel than NY-ESO-1. In some cases, LAGE-1 expression frequency isapproximately twice that of NY-ESO-1. Thus, in some exemplaryembodiments, identifying a suitable subject may comprise first screeningan HLA-A*0201 positive cancer patient for expression of LAGE-1, followedby further screening of the HLA-A*0201 positive, LAGE-1 positive patientfor expression of NY-ESO-1.

In some embodiments, a specific dosage regimen includes administering toa subject in need thereof a modified cell of the present disclosure. Inan exemplary embodiment, a specific dosage regimen includesadministering to a subject in need thereof, e.g., autologous T cellstransduced with a lentiviral vector to express NY-ESO-1 andelectroporated with Cas9 and guide RNA to disrupt expression ofendogenous receptor (e.g., TRAC, TRBC and/or PD1).

In some embodiments, a specific dosage regimen of the present disclosureincludes a lymphodepletion step prior to the administration of themodified T cells. In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide and/or fludarabine. In anexemplary embodiment, for a subject having multiple myeloma, the subjectreceives lymphodepleting chemotherapy prior to the administration of themodified T cells. In an exemplary embodiment, for a subject havingmultiple myeloma, the subject receives lymphodepleting chemotherapyincluding about 1.5 g/m² of cyclophosphamide by intravenous infusion. Inan exemplary embodiment, for a subject having multiple myeloma, thesubject receives lymphodepleting chemotherapy including about 1.5 g/m²of cyclophosphamide by intravenous infusion about 2 days (±1 day) priorto administration of the modified T cells. In an exemplary embodiment,for a subject having a sarcoma or melanoma, the subject receiveslymphodepleting chemotherapy prior to the administration of the modifiedT cells. In an exemplary embodiment, for a subject having a sarcoma ormelanoma, the subject receives lymphodepleting chemotherapy includingabout 300 mg/m² of cyclophosphamide and 30 mg/m² fludarabine byintravenous infusion. In an exemplary embodiment, for a subject having asarcoma or melanoma, the subject receives lymphodepleting chemotherapyincluding about 300 mg/m² of cyclophosphamide and 30 mg/m² fludarabineby intravenous infusion at 4 days, at 3 days, and at 2 days prior toadministration of the modified T cells.

In some embodiments, the methods of the invention involve selecting andtreating a subject having failed at least one prior course of standardof cancer cancer therapy. For example, a suitable subject may have had aconfirmed diagnosis of relapsed refractory multiple myeloma, melanoma,synovial sarcoma, or myxoid/round cell liposarcoma (MRCL).

In an exemplary embodiment, a suitable subject is a subject having had aconfirmed prior diagnosis of active myeloma as defined by IMWG criteria.In one embodiment, a suitable subject has relapsed or refractory diseaseafter either one of the following: 1) at least 3 prior regimens, whichmust have contained an alkylating agent, proteasome inhibitor, andimmunomodulatory agent (IMiD); or 2) at least 2 prior regimens if“double-refractory” to a proteasome inhibitor and IMiD, defined asprogression on or within 60 days of treatment with these agents. In someembodiments, induction therapy, stem cell transplant, and maintenancetherapy, if given sequentially without intervening progression, shouldbe considered as 1 “regimen.” In some embodiments, a suitable subject isat least 90 days since autologous stem cell transplant, if performed. Insome embodiments, a suitable subject may experience toxicities fromprior therapies, with the exception of alopecia or peripheral neuropathyattributable to bortezomib. In such cases, toxicities from priortherapies must have recovered to grade ≤2 according to the CTC 4.0criteria or to the subject's prior baseline. In one embodiment, asuitable subject has measurable disease per IMWG criteria on studyentry, which must include at least 1 of the following: 1) serum M-spike0.5 g/dL (patients with IgA myeloma in whom serum proteinelectrophoresis may be deemed unreliable, due to co-migration of normalserum proteins with the paraprotein in the beta region, may beconsidered eligible as long as total serum IgA level is elevated abovenormal range); 2) 24 hr urine M-spike 200 mg; 3) involved serum freelight chain (FLC) 50 mg/L with abnormal ratio; 4) measurableplasmacytoma on exam or imaging; 5) bone marrow plasma cells 20%.

In an exemplary embodiment, a suitable subject is a subject having had aconfirmed prior diagnosis of melanoma, progressed after at least 2therapy lines, and/or has had measurable disease per RECIST 1.1 in orderto allow assessment of an anti-tumor response. In an exemplaryembodiment, a suitable subject is a subject having had a confirmed priordiagnosis of synovial sarcoma or MRCL, proven metastatic disease orsurgically inoperable local recurrence that have failed first linetreatment, and/or has measurable disease per RECIST 1.1 in order toallow assessment of an anti-tumor response.

In some embodiments, a suitable subject is a subject that has an ECOGperformance status of 0-2.

In an exemplary embodiment, a suitable subject is a subject havingdocumented NY-ESO-1 expression on tumor tissue. In an exemplaryembodiment, a suitable subject is a subject that is HLA-A*201 positive.

In some embodiments, a suitable subject is a subject that has adequatevital organ function as defined by: 1) serum creatinine ≤2.5 orestimated creatinine clearance ≥30 ml/min and not dialysis-dependent; 2)absolute neutrophil count 1000/μl and platelet count ≥50,000/μl(≥30,000/μl if bone marrow plasma cells are ≥50% of cellularity for MMpatients); 3) SGOT ≤3× the upper limit of normal and total bilirubin≤2.0 mg/dl (except for patients in whom hyperbilirubinemia is attributedto Gilbert's syndrome); and/or 4) left ventricular ejection fraction(LVEF) 45%, wherein the LVEF assessment is performed within 8 weeks ofenrollment.

J. Pharmaceutical Compositions and Formulations

Also provided are populations of immune cells of the invention,compositions containing such cells and/or enriched for such cells, suchas in which cells expressing the recombinant receptor make up at least50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore of the total cells in the composition or cells of a certain typesuch as T cells or CD8+ or CD4+ cells. Among the compositions arepharmaceutical compositions and formulations for administration, such asfor adoptive cell therapy. Also provided are therapeutic methods foradministering the cells and compositions to subjects, e.g., patients.

Also provided are compositions including the cells for administration,including pharmaceutical compositions and formulations, such as unitdose form compositions including the number of cells for administrationin a given dose or fraction thereof. The pharmaceutical compositions andformulations generally include one or more optional pharmaceuticallyacceptable carrier or excipient. In some embodiments, the compositionincludes at least one additional therapeutic agent.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. A “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical formulation, otherthan an active ingredient, which is nontoxic to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative. In some aspects, thechoice of carrier is determined in part by the particular cell and/or bythe method of administration. Accordingly, there are a variety ofsuitable formulations. For example, the pharmaceutical composition cancontain preservatives. Suitable preservatives may include, for example,methylparaben, propylparaben, sodium benzoate, and benzalkoniumchloride. In some aspects, a mixture of two or more preservatives isused. The preservative or mixtures thereof are typically present in anamount of about 0.0001% to about 2% by weight of the total composition.Carriers are described, e.g., by Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecells, preferably those with activities complementary to the cells,where the respective activities do not adversely affect one another.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended. Thus, in some embodiments,the pharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and/or vincristine. Thepharmaceutical composition in some embodiments contains the cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. The desired dosage can bedelivered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, the cellpopulations are administered parenterally. The term “parenteral,” asused herein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecells are administered to the subject using peripheral systemic deliveryby intravenous, intraperitoneal, or subcutaneous injection. Compositionsin some embodiments are provided as sterile liquid preparations, e.g.,isotonic aqueous solutions, suspensions, emulsions, dispersions, orviscous compositions, which may in some aspects be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyoi (for example, glycerol, propylene glycol, liquid polyethyleneglycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can contain auxiliary substancessuch as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. It will be readily apparent to those skilled in the art thatother suitable modifications and adaptations of the methods describedherein may be made using suitable equivalents without departing from thescope of the embodiments disclosed herein. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the present invention. All such modifications areintended to be within the scope of the claims appended hereto. Havingnow described certain embodiments in detail, the same will be moreclearly understood by reference to the following examples, which areincluded for purposes of illustration only and are not intended to belimiting.

EXAMPLES Example 1: Manufacture of NY-ESO-1 TCR Autologous T Cells withDisrupted Expression of Endogenous TRAC, TRBC, and PDCD1 (NYCE T Cells)

The disruption conditions for the triple knock-out system in which TRAC,TRBC and PDCD1 genes were targeted using Cas9 RNA and guide RNA (gRNA)specific for these genes were tested in vitro. PBMCs from two normaldonors were isolated from whole blood by Ficoll extraction, and T cellswere isolated from PBMCs by negative selection using the Pan T CellIsolation Kit II (Miltenyi Biotec). Isolated T cells were stimulatedovernight with Dynabeads Human T-Expander CD3/CD28 beads at a 3:1bead:cell ratio. T cells were then transduced with lentivirus vector(LV) encoding NY-ESO-1 TCR at a virus concentration adjusted to an MOIof 1 and incubated overnight. On Day 3 after LV transduction, CD3/CD28beads were removed and the pre-activated T cells were electroporatedwith Cas9 RNA followed by electroporation with three differentconcentrations of gRNA (H, M, and L) specific for the TCRα, TCRβ, andPD1 on Day 4. T cell expansion (FIG. 4 ), efficacy of gene disruption(FIGS. 5-8 ), cell functionality (FIGS. 9-11 ) and long term expansion(FIGS. 12 and 13 ) were tested.

Following LV transduction and Cas9 mRNA and gRNA electroporation, cellswere diluted with fresh media every other day during expansion tomaintain optimal growth. Total cell numbers were measured at multipletime points and used to determine the fold-expansion of T cells. Datagraphed in FIG. 4 indicate that cells from both donors showed adose-dependent decrease in T cell expansion as a function of increasingconcentrations of gRNAs.

Referring to FIG. 4 , T cells were isolated from normal, healthy donors(Donor 1-ND307 and Donor2-ND422) and transduced with (solid shapes) orwithout (open circles) NY-ESO-1 LV at MOI=1. On Day 3, cells wereelectroporated with Cas9 RNA and on Day 4 cells were electroporated withgRNA at 3 different doses: L (Low, solid triangles), M (Medium/Standard,solid circle) and H (High, solid diamond). Cell electroporation wasperformed at a concentration range of 1-3×10⁸/mL. Viable cells werecounted on Day 3, 10, and 12 (x-axis) of culture and T cell foldexpansion was calculated (y-axis).

To determine the efficiency of Cas9 gene editing of TCR^(endo), the Tcells were evaluated by flow cytometry for cell surface expression ofNY-ESO-1 TCR and endogenous TCR (FIGS. 5 and 6 ) on culture day 11.Cells were stained with mAb specific to the VP8 (binding to NY-ESO-1TCR) and CD3 (FIG. 5 ) or with a fluorescently labeled HLA-A2 dextramerfolded around the NY-ESO-1 epitope (FIG. 6 ).

Analysis was done by first gating on cells with a lymphocyte scatterprofile and then measuring expression of CD3 and VP8. The resultsshowed, for both donors, a high level of NY-ESO-1-specific TCRexpression was maintained across a range of gRNA concentrations, withNY-ESO-1 TCR expression ranging between 45-49% for one donor and 60-62%for the other donor T cells. Endogenous TCR gene editing was observed in46% to 90% of untransduced T cells and was dependent on the gRNAconcentration. Extrapolating from the data, at the medium dose of gRNA,70% of the NY-ESO-1 TCR expressing cells would lack endogenous TCR (FIG.5 ).

Referring to FIG. 5 , T cells from Day 11 expansion cultures from twodonors (ND307 and ND422) of NY-ESO-1 transduced cells, electroporatedwith three different concentrations (High, Medium, Low dose) of gRNAsfor TCRα, TCRβ, and PD1 were analyzed by flow cytometry for CD3 and VP8surface expression. Analysis was done by first gating on a lymphocytescatter profile followed by analysis of CD3 (surrogate for TCRexpression; x-axis) and VP8 (binds to NY-ESO-1 TCR; y-axis). Both donorsexhibited low level background staining with the anti-VP8 mAb; hence,this mAb reliably identified NY-ESO-1 TCR-expressing transgenic T cells.

The frequency of NY-ESO-1-specific T cells was confirmed by flowcytometry using a fluorescently labeled HLA-A2/NY-ESO-1 dextramer (FIG.6 , solid bars). Surface expression of the transduced TCR chains wasdramatically enhanced with increasing concentrations of gRNAs indicatingthat antigen recognition by this TCR was enhanced when the endogenousTCR chains were knocked out, as previously reported for other transgenicTCRs. At the high concentrations of gRNA the % of T cells expressingNY-ESO-1 TCR was equivalent to that seen in FIG. 5 when NY-ESO-1expression was assessed by VP8 mAb. Furthermore, transduction efficiencywas assessed using a validated WPRE (for the Woodchuck Hepatitis VirusPosttranscriptional Regulatory Element) qPCR, (part of the lentiviralNY-ESO-1 TCR vector). This analysis revealed one or less copies ofintegration of the NY-ESO-1 viral vector per cell (FIG. 6 , barsoutlined in grey and right side y-axis).

Referring to FIG. 6 , T cells from Day 11 expansion cultures from donorND422 of NY-ESO-1 transduced cells, electroporated with three differentconcentrations (high, medium, low) of gRNAs for TCRα, TCRβ, and PD1 wereanalyzed by flow cytometry using fluorescently conjugated HLA-A*0201dextramers folded around the NY-ESO-1 peptide SLLMWITQC (grey filledbars; SEQ ID NO:1) and also by qPCR specific for the WPRE (grey outlinedbars). Shown is the expression of this TCR in total T cells.

To determine the efficiency of PD1 gene editing, the same Day 11cultures were restimulated with CD3/CD28 beads for 3 days to induce PD1expression (FIG. 7 ). Without re-stimulation, NY-ESO-1 transduced butnot edited cells had a background PD1 expression of 14.7%. Withstimulation, in the absence of gene editing for PD1 in either LVtransduced (No CRISPR) or non-LV transduced (No TD No CRISPR), greaterthan 95% of CD3 positive cells were induced to express PD1. Thepercentage of LV transfected T cells electroporated with gRNA andlacking PD1 expression dropped to 35%, 57%, and 74% for L, M, and H gRNAconcentrations respectively. Thus, at the medium/standard dose of gRNA,57% of the NY-ESO-1⁺ T cells were deficient in the ability to expressPD1 (PD1⁻).

Referring to FIG. 7 , T cells from Day 11 expansion culture of NY-ESO-1transduced cells electroporated with three different concentrations(High, Standard, Low) of gRNAs for TCRα, TCRβ, and PD1 were analyzed byflow cytometry. T cells that were not transduced with NY-ESO-1lentiviral vector (Non Transduced) but electroporated with the 3concentrations of gRNAs were also prepared. Control cells that were notelectroporated were prepared for each of the transduced andnon-transduced cell populations. These cell populations wererestimulated with CD3/CD28 beads (Re-stim) to induce PD1 expression orwere left unstimulated (No Re-stim). Three days later, all cellpopulations were surface stained with mAb specific for CD3 and PD1.Analysis was done by first gating on a lymphocyte scatter profile andthen analyzing for CD3 expression (surrogate for TCR expression; x-axis)and PD1 (y-axis).

In addition to flow cytometry analysis for protein expression, thefrequency of targeted gene disruption was measured by amismatch-selective surveyor nuclease assay on DNA amplified from thecells on day 12 of culture (FIG. 8 ). For each donor, a dose responsewas observed correlating the % of gene editing with the concentration ofgRNA used. This was observed for all three genes: TCRα, TCRβ, and PD1though the extent of gene editing varied by donor. This assaydemonstrated efficient disruption for all the genes in donor 307. Fordonor 422 T cells, the disruption of TCRα was not optimal, however, theother genes were efficiently disrupted.

Referring to FIG. 8 , primer sets for each gene (TCRα, TCRβ, and PD1)were used in regular, singular PCR reactions. PCR products were treatedwith endonuclease and run on polyacrylamide gels (left panel). Theintensity of the intact and digested bands were measured and % geneknockout was calculated for both donors (right pantel:dark greybars=ND307; light grey bars=ND422). % gene knockout was calculated forfour concentrations of gRNA (high, medium, low, none) for cells preparedas described in FIG. 4 above.

Functionality of NY-ESO-1 transduced T cells with TCR^(endo) and PD1gene edited was measured by the cells ability to degranulate (FIG. 9 ),release IL-2 and IFNγ (FIG. 10 ), and lyse (FIG. 11 ) upon co-culturewith tumor cell lines expressing NY-ESO-1.

To determine the ability of NY-ESO-1 transduced cells to degranulate inresponse to Nalm6-HLA-A2-expressing NY-ESO-1 transfected tumor cell line(Nalm6-ESO), T cells from the above expansion cultures, from two donors,were assessed by flow cytometry for the appearance of expression ofCD107a on the surface of the T cells. For both donors, only in theNY-ESO-1 transduced T cells was degranulation observed via surfaceexpression of CD107a. The percentage of T cell degranulation wasincreased in the TCR^(endo) and PD1 gene edited cells and was dosedependent (FIG. 9 ).

Referring to FIG. 9 , T cells prepared as described in FIG. 4 wereco-incubated with a NaIm6-HLA-A2-expressing NY-ESO-1 transfected tumorcell line (NamI6-ESO) or a control cell line without NY-ESO-1 expression(NamI6). Cells were surface stained with mAbs specific for CD8 andCD107a and analyzed by flow cytometry.

Next, the ability of NY-ESO-1 transduced T cells with TCR^(endo) and PD1gene edited to release IL-2 and IFNγ in the presence or absence ofNY-ESO-1 presenting tumors was assessed. NY-ESO-1 transduced T cellsreleased IFNγ and IL-2 in response to Nalm6-HLA-A2-expressing NY-ESO-1transfected tumor cell line (Nalm6-ESO) and to a NY-ESO-1 expressingmelanoma tumor cell line, but not when incubated with non-NY-ESO-1expressing tumor (Nalm6). In addition, there was a clear dose dependenceof cytokine production in response to the amount of gene editing.Cytokine production was superior for TCR^(endo) and PD1 gene editedcells compared to NY-ESO-1 transduction alone for both donors whichindicates a functional advantage to gene editing of TCR^(endo) and PD1(FIG. 10 ).

Referring to FIG. 10 , NY-ESO-1 transduced T cells (TD) ornon-transduced (No TD) cells prepared as described in FIG. 4 wereco-incubated overnight with a Nalm6-HLA-A2-expressing NY-ESO-1transfected tumor cell line (Nalm6-ESO) or a control cell line withoutNY-ESO-1 expression (Nalm6) or with a melanoma cell line expressingNY-ESO-1 (624mel). Culture supernatant was collected and IFNγ or IL-2measured by ELISA. The ability of NY-ESO-1 transduced T cells in theabsence of gene editing (TD NO KO) to release cytokine in response totumor was also evaluated. T cells from expansion cultures from twodonors (307 and 422) were tested.

To determine the ability of NY-ESO-1 transduced or non-transduced Tcells with or without gene editing of TCR^(endo) and PD1 to kill aHLA-A2 NY-ESO-1 tumor cell line, a luminescence assay of tumor specificlysis was performed. Nalm6-ESO-CBG cells were resuspended at 1×10⁵cells/mL and incubated with different ratios of T cells (e.g. 30:1,15:1, etc.) overnight at 37° C. 100 μl of the mixture was transferred toa 96 well white luminometer plate, 100 ul of substrate was added and theluminescence was immediately determined. Gene edited T cells transducedwith NY-ESO-1 showed superior ability to lyse the tumor cell targetacross all E:T ratios above either non-transduced cells (No TD) orNY-ESO-1 transduced cells without gene editing (TD NO KO) (FIG. 11 ).

Referring to FIG. 11 , NY-ESO-1 transduced T cells (TD) ornon-transduced (No TD) with (solid triangles) or without (solid circlesor X) gene editing for TCR^(endo) and PD1 were co-cultured withNalm6-HLA-A2-expressing NY-ESO-1 transfected tumor cell line(NamI6-ESO). Tumor cell lysis was determined by luminescence assay andexpressed as tumor specific lysis (y-axis) as a function of the effectorto target ratio (E:T; x-axis). T cells from Day 12 cultures from donor307 were tested. Results are reported as percent killing based onluciferase activity in wells with tumor, but no T cells. (%killing=100−((RLU from well with effector and target cellcoculture)/(RLU from well with target cells)×100)).

Without being bound by any theory, these data indicate that NY-ESO-1 TCRtransduced and triple edited cells exert superior functional activitycompared to the NY-ESO-1 TCR transduced cells only and that thedisruption of the targeted genes does not negatively affect theirfunctional potential.

To determine if TCR^(endo) and PD1 gene editing introduces off-targetmutations that influence cell proliferation, Day 12 T cells were placedin culture without additional stimulation. After initial expansion, allconditions plateaued or contracted by day 42. Viability either remainedstable or decreased. Cell size steadily decreased in all conditions(FIG. 12 ). The populations of NY-ESO-1 transduced T cells withTCR^(endo) and PD1 gene editing are expected to continue to contractsimilarly to non-modified cells. These data demonstrate the absence ofTCR^(endo) and PD1 gene editing-associated transforming mutations.

Referring to FIG. 12 , NY-ESO-1 transduced T cells with TCR^(endo) andPD1 gene editing from Day 12 cultures were placed in long term culture.Initial long-term cultures were seeded with 2×10⁷ cells per condition inRPMI10 medium with IL2 (100 IU/ml) at 1×10⁶ cells/mL. Cell number, cellviability and cell size were measured throughout the culture on the daysindicated. Cells were fed every other day with RPM110 mediumsupplemented with IL2.

Surface expression of the NY-ESO-1 TCR was again assessed at day 38using the aforementioned HLA-A2/NYESO1 dextramer staining. The data(FIG. 13 ) demonstrated that high level surface expression of this TCRwas maintained throughout culture, and, again, both CD4+ and CD8+ Tcells showed similarly high frequencies of NYESO1-specific TCRexpression with the same titration effect as shown in FIG. 6 .

Referring to FIG. 13 , cell populations at the end of long term cultures(day 38 in FIG. 9 ) were analyzed by flow cytometry for NY-ESO-1 TCRexpression using the method described in FIG. 6 . The graph represents %NY-ESO-1 expressing T cells (y-axis) as a function of gRNA concentration(none, low, medium, high; x-axis) for both donor T cells.

Without being bound by any theory, these data demonstrated that thetriple CRISPR editing system combined with LV transduction of T cellswas successful in redirecting T cells and provided a gain in functionpotential superior to that of LV transduced T cells only. The NY-ESO-1TCR expressing T cells edited at the endogenous TCR and PD-1 loci wereable to expand, secrete cytokine, degranulate and lyse antigen specifictargets in vitro supporting their proposed clinical use.

Prophetic Example 1: Manufacture of Clinical NY-ESO-1 TCR Autologous TCells with Disrupted Expression of Endogenous TRAC, TRBC, and PDCD1(NYCE T Cells)

NYCE T cells are autologous NY-ESO-1-redirected and CRISPR TCR^(endo)and PD1 edited T cells. Autologous T cells will be engineered using alentiviral vector to express a TCR with specificity for NY-ESO-1 peptide(SLLMWITQC; SEQ ID NO:1) in complex with HLA-A*0201 in which theendogenous TCR (a and β chains) and PD1 have been disrupted. Withoutbeing bound to any theory, this is expected to enhance the potency ofthe transduced T cells toward NY-ESO-1 expressing targets since a) thenegative checkpoint regulator PD1 is knocked out and b) surfacedimerization of the transgenic TCRs is achieved in the absence ofTCR^(endo) expression. In addition, since the endogenous and introducedTCR chains can dimerize and create novel, unwanted, specificities, theknockout of the TCR^(endo) will enhance the safety of NYCE T cells.

The NYCE T cells will be manufactured in the Clinical Cell and VaccineProduction Facility (CVPF) at the University of Pennsylvania. At the endof cell culture, the cells are cryopreserved in infusible cryomedia.Each bag will contain an aliquot (volume dependent upon dose) ofcryomedia containing the following infusible grade reagents (% v/v):31.25% plasmalyte-A, 31.25% dextrose (5%), 0.45% NaCl, 7.5% DMSO, 1%dextran 40, 5% human serum albumin. NYCE T cells will be administered asa single infusion. The target dose is 1×10⁸ total cells/kg.

Absorption, distribution and metabolism. Lymphocytes have complextrafficking and survival kinetics, and after adoptive transfer severalfates have been demonstrated: 1) margination; 2) exit from theperipheral blood and trafficking to lymphoid tissues; and 3) death byapoptosis. Following an intravenous dose, retrovirally modified andadoptively transferred T cells have been shown to persist in thecirculation for at least 10 years in immunodeficient SCID patients dueto the replicative competence of T cells. Human CD8 CTLs have anelimination half-life from the peripheral blood of about 8 daysfollowing intravenous infusion, and this increases to about 16 days whenlow doses of IL-2 are given. In patients with HIV infection, the meanhalf-life of lentivirally modified CD4 T cells in the circulation of 5patients following a single infusion was 23.5 (±7.7) days in patients.Adoptively transferred human T cells have been shown to traffic to tumorand secondary lymphoid tissues. CD19 CAR T cells (CART19) have persistedbeyond 5 years in patients with chronic lymphocytic leukemia and acutelymphocytic leukemia.

Drug interactions. Without being bound by any theory, NYCE T cells areexpected to retain many of the properties of natural T cells. As such,they will be expected to be susceptible to immunosuppressive agents suchas corticosteroids, calcineurin inhibitors such as cyclosporine andtacrolimus, anti-metabolite agents including methotrexate andmycophenolate mofetil, mTOR inhibitors such as sirolimus and everolimus,and lymphodepleting antibodies including, alemtuzumab, daclizumab, anddenileukin diftitox. Lymphocytes are also susceptible to cytotoxic andchemotherapeutic agents that are commonly administered for hematologicmalignancies such as cyclophosphamide and fludarabine.

Immune elimination. Without being bound by any theory, it is possiblethat the engineered NYCE T cells may be immunogenic, and that thepatients will have an immune response directed against the cells,resulting in cell elimination. Immune responses may be directed towardsthe NY-ESO-1 transgenic TCR, however this was not observed in previousstudies. It is also possible that immune responses may be directedagainst the edited gene products of the endogenous TCR or against editedPD1. Products from the CRISPR/Cas9 editing process may be partiallytranslocated, not be displayed on the cell surface and yet trigger animmune response. Cas9 is a protein derived from Streptococcus; this isexpected to be immunogenic. Since the gene-edited cells will undergoextensive proliferation following the initial editing, the Cas9 proteinproduct is expected to be present at exceedingly low or undetectablelevels in the infused cell product. Immunogenicity of the infusedproduct will be monitored as a secondary correlative analysis. If it isfound that the T cells have prolonged engraftment, then immunogenicityis not a major issue.

Rationale for lymphodepletion. Adoptive immunotherapy strategies may beable to capitalize on homeostatic T cell proliferation, a finding thatnaive T cells begin to proliferate and differentiate into memory-like Tcells when total numbers of naive T cells are reduced below a certainthreshold. Lymphodepletion reduces or eliminates regulatory T cells andother competing elements of the immune system that act as “cytokinesinks,” enhancing the availability of cytokines such as IL-7 and IL-15that promote expansion of adoptively transferred T cells. Thishypothesis has been tested clinically in patients with metastaticmelanoma refractory of conventional treatments. The patients received alymphodepleting conditioning regimen consisting of cyclophosphamide (60mg/kg×2 days) and fludarabine (25 mg/m²×5 days) prior to adoptivetransfer of T cells. Treated patients with myeloma and lymphoma afterlymphodepleting chemotherapy have been observed to exhibit improvedengraftment in lymphoma, as well as in randomized cohorts of myeloma andneuroblastoma patients. In this experiment NYCE T cells will beadministered into subjects that are rendered lymphopenic as a result ofcytotoxic chemotherapy. The chemotherapy regimen used forlymphodepletion will be disease specific.

Predicted cell populations generated during product manufacture. Basedon the lentiviral gene transfer of the NY-ESO-1 TCR and the CRISPR/Cas9editing targeting of PDCD1, TRAC and TRBC genes, there are 16 possiblepopulations of autologous T cells that can be generated during themanufacturing process (Table 2).

TABLE 2 NYESO1- NYESO1+ NYESO1- NYESO1+ NYESO1- NYESO1+ NYESO1- NYESO1+TCR WT TCR WT TCR A-B+ TCR A-B+ TCR B-A+ TCR B-A+ TCR A-B- TCR A-B- PD1WT 1 2 3 4 5 6 7 8 PD1- 9 10 11 12 13 14 15 16 CD3 1 2 3 4 5 6 7 8 CD3neg CD3 9 10 11 12 13 14 15 16 CD3+ Tetramer 1 2 3 4 5 6 7 8 Tetramerneg Tetramer 9 10 11 12 13 14 15 16 Tetramer +

These 16 cell types reduce down to 6 possible cell surface phenotypes asdetected by staining with NY-ESO-1 tetramer, anti-CD3 and anti-PD-1antibodies. Within those 6 possible cell surface phenotypes, there aredifferent potential safety and efficacy profiles based on NY-ESO-1 TCR,PD-1, TCRα and TCRβ expression. A detailed breakdown of these profilesis outlined in Table 3.

TABLE 3 Cell Cell surface surface staining Gene status stain- NY-(intact +) or ing ESO- (disruption -) Cell Predicted safety and efficacyprofile 1 CD3 PD1* TCRα TCRß PD1 Pop'n profile 1 − + + + + + 1 Wild-typeT cells that were not modified during manufacturing. Safety is expectedwhile efficacy is not. 2 − + − + + − 9 T cells with PD-1 disruption thatretains endogenous TCR. These cells are anticipated to have a lowtoxicity risk based on data from systemic therapy with PD-1 antagonists,however they could mediate autoreactivity. These cells may facilitate″antigenic spreading″ of an antitumor response. 3 − − + − + + 3 T cellswith endogenous TCR + − + 5 disruption. These cells are − − + 7 expectedto be rare, have low potential for toxicity and should not persist afteradoptive transfer. 4 − − − − + − 11 T cells with endogenous TCR + − − 13and PD-1 disruption; expected to − − − 15 be rare, have low potentialfor toxicity and should not persist after adoptive transfer.5 + + + + + + 2 NY-ESO-1 specific T cells without gene disruption.Previously tested in patients with multiple myeloma with demonstratedsafety. − + + 4 NY-ESO-1 specific T cells with + − + 6 endogenous TCRdisruption. Expected enhanced anti-tumor activity compared to cellpopulation 2, have reduced, but not absent potential for mispairing andsusceptible to exhaustion by PD-1 expression. − − + 8 NY-ESO-1 specificT cells with endogenous TCR disruption. Expected enhanced anti-tumoractivity compared to population 2. Absent potential for mispairing, butsusceptible to exhaustion by PD-1 expression. 6 + + − + + − 10 NY-ESO-1specific T cells with PD-1 disruption and retained endogenous TCR.Expected anti-tumor efficacy and less susceptible to exhaustion by PD- 1expression − + − 12 NY-ESO-1 specific T cells with + − − 14 endogenousTCR and PD-1 − − − 16 disruption. Expected reduced potential formispairing. May have enhanced effector function. Expected to be lesssusceptible to exhaustion by PD-1 expression. *after stimulation

Population #1 is wild type T cells that escaped transduction or editingduring manufacturing. These autologous T cells are expected to be safeand have no antitumor activity based on previous trials. Population #2are T cells with transgenic TCR and the retained endogenous TCR. Thesecells have been tested clinically and found to be safe and haveantitumor efficacy in sarcoma, melanoma and myeloma. Population #9 are Tcells with PD-1 disruption that retain endogenous TCR. These cells areanticipated to have a low toxicity risk based on data from systemictherapy with PD-1 antagonists, but may mediate autoreactivity. Withoutbeing bound by any theory it is possible that these cells may facilitate“antigenic spreading” of an antitumor response directed against tumorneo-epitopes. Populations #3, 5, 7, 11, 13, and 15 are T cells withendogenous TCR disruption. These cells are expected to be rare, have lowpotential for toxicity and should not persist after adoptive transferbecause TCR signals are required for T cell survival in experimentalsettings. Populations #10, 12, 14, and 16 are NY-ESO-1 specific T cellswith PD-1 disruption. These cells may have enhanced effector functionand are expected to be less susceptible to exhaustion by PD-1expression. A major rationale for this experiment is that it is acompetitive repopulation experiment: the bulk cells will be infused withthe expectation that the immune competition and tumor microenvironmentwill select the “winners,” testing the hypothesis that T cells withNY-ESO-1 specificity and disrupted PD1 may have enhanced function.

Prophetic Example 2: Administration of NYCE T Cells to Patients

NYCE T cells are autologous NY-ESO-1-redirected and CRISPR TCR^(endo)and PD1 edited T cells. Autologous T cells will be engineered using alentiviral vector to express a TCR with specificity for NY-ESO-1 peptide(SLLMWITQC; SEQ ID NO:1) in complex with HLA-A*0201 in which A pilottrial will be done to determine if the infusion of autologous NYCE Tcells (transduced to express NY-ESO-1 TCR and lacking endogenous TCR andPD-1) is safe. The primary goals of the trial are to determine thesafety of NYCE T cells in relapsed and/or refractory multiple myeloma(MM), sarcoma and melanoma patients. The protocol consists of an openlabel pilot study. The general protocol schema is shown in FIG. 14 .

As part of informed consent, patients will be asked for permission totest their HLA-A*201 status and their tumor for expression of NY-ESO-1.HLA-A*201 status will be confirmed first. Patients who are HLA-A*201positive will then have their tumor tissue tested for NY-ESO-1expression. Expression testing can be performed on historical samples ifavailable (see Section 6.1 for additional details). Patients who areHLA-A*201+ and express NY-ESO-1 in their tumor samples may proceed tothe next step and be presented the main informed consent form to undergoadditional screening.

At study, entry subjects will undergo routine laboratory and imagingassessment of their disease. Eligible subjects will undergo steady-stateapheresis to obtain large numbers of peripheral blood mononuclear cells(PBMC) for manufacturing. Cryopreserved historical apheresis productscollected from the patient prior to study entry are usable formanufacturing if collected at an appropriately certified apheresiscenter and the product meets adequate mononuclear cell yields. If ahistorical apheresis product is not available, an apheresis procedurewill be scheduled for cell procurement after study entry. The T cellswill be purified from the PBMC, transduced with lentiviral vector todeliver the recombinant NY-ESO-1 TCR and electroporated with CRISPR/Cas9complexes, expanded in vitro and then frozen for future administration.The number of subjects who have inadequate T cell collections, expansionor manufacturing compared to the number of subjects who have cellssuccessfully manufactured will be recorded.

The manufacturing process and product release will take 4-5 weeks fromtime of apheresis until NYCE T cells are ready for infusion. Patientsmay receive additional specific anti-tumor therapy to keep their diseasein check during this interval, at their physician's discretion. Patientsmust be off all therapy, however, during the 2 weeks prior to theplanned infusion date.

Up to 18 evaluable subjects: 6 myeloma subjects, 6 sarcoma subjects and6 melanoma subjects will be enrolled. Patients with multiple myeloma andsarcoma will be targeted for enrollment first, with the plan to expandenrollment to include melanoma patients once initial safety isestablished. Subjects will be enrolled serially. Infusions of the first2 subjects in each disease indication will be staggered by 28 days toallow for observation of adverse events.

In the event that the clinical target dose is not met for a particularsubject, the subject has the option of undergoing a second apheresis andmanufacturing run. Alternatively, the subject can be treated with theavailable dose that is lower than the target dose. In this case, thesubject will be evaluated for primary endpoints (safety andfeasibility), but not for efficacy secondary endpoints. Subjects who donot receive NYCE T cells will be replaced.

All subjects will have evaluations, physical exams, and blood tests toassess safety and engraftment/persistence of the NYCE T cells at regularintervals.

Disease assessment will be performed at Day +28 and at 3 monthspost-infusion for all indications.

Formal myeloma response assessments will be made according toInternational Myeloma Working Group (IMWG) criteria at Days +14 and +28,then monthly until Day +180, then every 3 months thereafter, up to 2years. Subjects with MM will undergo bone marrow aspirates/biopsies toassess the bone marrow plasma cell burden at day +28 and Month 3.

Melanoma response assessment will be made using (i) clinical exam forvisible cutaneous tumors, (ii) radiological imaging (CT scan) withRECIST 1.1 criteria, (iii) pathological criteria for resected tumortissue.

Sarcoma response assessment will be made using radiological imaging (CTscan) with RECIST 1.1 criteria. CT scans of the chest for synovialsarcoma and CT scans of the chest/abdomen/pelvis for MRCL. If there islocally unresectable disease in the extremity or pelvis, it will beassessed by MRI with/without gadolinium contrast (no lab studiesapplicable for tumor progression).

Subsequent biopsy collections (tumor, bone marrow) will be performed atDay 28 and as clinically indicated at the discretion of the treatingphysician; samples from these biopsies may be provided to TCSL forcorrelative studies.

Upon discontinuation from the primary follow-up phase, subjects willenter long-term follow-up for up to 5 years from their NYCE cellinfusion. During long-term follow-up, subjects will be monitored fordelayed adverse events that may be associated with the administration ofthe NYCE T cells, as well as T cell persistence and disease progression(as applicable).

Example 2: PD1-CD28 Switch Receptor Boosts NY-ESO-1 TCR T Cell FunctionThrough Evading Suppression from Tumor Microenvironment

Safety and potency are two major requirements for an effective adoptiveimmunotherapy of cancers using genetically TCR engineered T cells.Current therapies of treating cancers using TCR engineered T cells,especially for solid tumors, are limited by poor T cell function andpoor T cell persistence, majorly due to tumor microenvironment induced Tcell hypofunction and exhaustion, as well as safety concerns due toeither mis-pairing of introduced TCR with endogenous TCR or usingaffinity enhanced TCRs that potentially cause un-predicted off targettoxicities.

High efficient multiplex gene disruption of both TRAC and TRBC in a wildtype NY-ESO-1 TCR transferred T cells using CRSIPR/CAS9 gene editing wasfound to improve antigen specific T cell functions both in vitro and invivo in tumor mouse models.

T cells transferred with 8F NY-ESO-1 TCR via electroporation werecharacterized (FIGS. 15A-15C). FIG. 15A shows CD3 and transferred TCR(vb8) expression of TRAC/TRBC disrupted CD3− T cells (CD3−) that wereelectroporated with RNAs for TCR alpha and beta of 8F NY-ESO-1 TCR (TCREP). FIG. 15B shows the lytic activity of 8F NY-ESO-1 TCR transferredTRAC/TRBC CRISPR/CAS9 disrupted CD3 negative T cells (TCR EP/CD3−),compared with the same TCR transferred non-CRISPR/CAS9 gene editedregular CD3 positive T cells (TCR EP/CD3+). FIG. 15C shows the level ofcytokine production of 8F NY-ESO-1 TCR transferred TRAC/TRBC CRISPR/CAS9disrupted CD3 negative T cells (TCR EP/CD3−), compared with the same TCRtransferred non-CRISPR/CAS9 gene edited regular CD3 positive T cells(TCR EP/CD3+).

Stress tests were performed for CRISPR gene editing of NY-ESO-1 TCRtransduced T cells (FIGS. 16A-16B). FIG. 16A shows the expression of TCR(vb8), CD3 and PD1 in the T cells transduced (TD) (or non-transduced (NOTD)) with 8F NY-ESO-1 TCR and subjected to different doses ofCRISPR/CAS9 RNA. FIG. 16B shows the expansion of the T cells that weresubjected to different doses of CRISPR/CAS9 RNA.

Referring to FIG. 17 , FIG. 17 shows the level of cytokine production ofthe NY-ESO-1/HLA-A2 positive cell lines Nalm6-ESO or 624mel stimulatedwith T cells that were transduced with 8F NY-ESO-1 TCR (TCR TD) andsubjected to TRAC/TRBC/PD1 disruption with different doses of CAS9/gRNA.

TRAC/TRBC disruption was examined across T cells transduced withdifferent TCRs (FIG. 18 ). FIG. 18 shows the transduced TCR expressionand tetramer staining of TRAC and TRBC disrupted T cells that weretransduced with either wild type NY-ESO-1 TCR (8F or 1G4) or affinityenhanced 1G4 NY-ESO-1 TCR for (Ly95).

FIG. 19 shows the level of cytokine production of differentNY-ESO-1/HLA-A2 positive cell lines (Nalm6-ESO, 624mel or A549-ESO)stimulated with TRAC and TRBC disrupted T cells that were transducedwith either wild type NY-ESO-1 TCR (8F or 1G4) or affinity enhanced 1G4NY-ESO-1 TCR (Ly95).

FIG. 20 shows the lytic activity of the TRAC and TRBC disrupted T cellsthat were transduced with either wild type NY-ESO-1 TCR (8F or 1G4) oraffinity enhanced 1G4 NY-ESO-1 TCR (Ly95) against the A549-ESO tumorline.

CRISPR/CAS9 disruption of TRAC and TRBC was found to improve thefunction of both wild type and affinity enhanced NY-ESO-1 TCR transducedT cells, while the specificity was only maintained for T cells with wildtype TCRs (FIGS. 21A-21B). FIG. 21A shows HLA-A2 expression of K562electroporated with different amount of HLA-A2 in vitro transcribed RNA.FIG. 21B shows CD107a expression of the TRAC and TRBC disrupted T cellsthat were transduced with either wild type NY-ESO-1 TCR (8F or 1G4) oraffinity enhanced 1G4 NY-ESO-1 TCR (Ly95) stimulated by theNY-ESO-1/HLA-A2 positive tumor lines (624mel and A549-ESO), theNY-ESO-1/HLA-A2 negative tumor line A549, and the NY-ESO-1 negative K562electroporated with different amounts of HLA-A2 RNA.

To further boost the in vivo anti-tumor activities of the NY-ESO-1 TCRtransferred T cells, a PD1 switch receptor, comprising the truncatedextracellular domain of PD1 and the transmembrane and cytoplasmicsignaling domains of CD28 (PD1-CD28) was co-introduced into TRAC andTRBC disrupted NY-ESO-1 T cells.

The NYESO-1 TCR and PD1-CD28 switch receptor were cloned into the samelentivirus vector. As such, the lentivirus vector contained sequencesfor NYESO-1 TCR alpha chain, NYESO-1 TCR beta chain, and a PD1-CD28switch receptor, as well as WPRE, cPPR, and a EF-1α promoter, as shownin FIG. 22 .

FIG. 22 shows a lentivirus vector map. As shown, the PD1-CD28 sequenceis separated from the NY-ESO-1 (8F) sequence by a F2A sequence asdescribed herein. The NY-ESO-1 (8F) alpha and beta chain sequences areseparated by an F-GS2-T2A sequence as described herein.

In vitro function tests of CRISPR/CAS9 gene edited, NY-ESO-1 TCR (8F)transduced T cells, with or without co-expressing the PD1-CD28 switchreceptor were performed (FIGS. 23A-23B). FIG. 23 shows cytokineproduction (FIG. 23A) and tumor specific lysis (FIG. 23B) aredramatically increased in TCR KO T cells.

Treatment of mice bearing solid tumors with PD1-CD28, NY-ESO-1 T cellsled to significant regression in tumor volume due to enhanced TILinfiltrating, decreased susceptibility to tumor-induced hypofunction,resistance of tumor derived suppression, such as from TGFβ, adenosine,IDO, hypoxia and Treg, compared to NY-ESO-1 T cells with TRAC/TRBCdouble disruption, or TRAC/TRBC/PD1 triple disruption (FIGS. 24A-25C).

A549.ESO.CBG was subcutaneously injected at 5×10⁶ cells/mouse at day 0.At day 9 tumor T cells injection (i.v.) at 1×10⁷ cells/mouse (FIG.24A-25B). FIG. 24A shows the BLI (bioluminescence imaging) that wasconducted weekly. FIG. 24B shows a graph of tumor size that was measuredone day prior to the T cell treatment, and weekly post-treatment. InFIG. 24B, NTD: non-transduced; 8F PD1 KO: 8F transduced T cells withdisrupted PD1; 8F DKO: 8F transduced T cells with disrupted TRAC andTRBC; 8F TKO: 8F transduced T cells with disrupted TRAC, TRBC and PD1;PD1CD28.8F DKO: 8F and PD1-CD28 switch receptor transduced T cells withdisrupted TRAC and TRBC.

A second set of mouse experiments were performed (FIGS. 25A-25C). FIG.25A shows a table of the experimental groups, with 10 mice/group. FIG.25B shows a timeline of the mouse experiments. FIG. 25C shows BLI(bioluminescence imaging) of the mice. FIG. 25D shows a graph of tumorsize that was measured one day prior to the T cell treatment, and weeklypost-treatment.

A screen was performed to identify effective gRNAs targeting TRAC, TRBC,and PD1 (FIGS. 26A-26C). FIGS. 26A-26C show a series of plotsillustrating TRAC (FIG. 26A), TRBC (FIG. 26E), and PD1 (FIG. 26C) genedisruption efficiency in T cells screened by electroporation ofCRISPR/CAS9 RNA for gRNAs targeting TRAC, TRBC, and PD1, respectively.

Off-Target detection by Guide-seq was performed (FIGS. 27A-27D). FIG.27A shows a table of samples' treatment. FIG. 27B shows the expressionof PD1 and CD3 in T cells of each sample. FIG. 27C shows the validationof capture of double-stranded oligodeoxynucleotides (dsODN) into DSBs.FIG. 27D shows a summary of the Guide-seq results for the off-targetsites. The percent off-target sites was: Group I guide RNAs: 1.7%; GroupII guide RNAs: 0.19%.

Additional disruption of Tim-3 for TRAC/TRBC double disrupted PD1-CD28NY-ESO-1 T cells showed synergistic effect of controlling large,established solid tumors in mice. A screen was performed to identifyeffective gRNAs targeting the immune checkpoint protein TIM-3 (FIGS.28A-28D). FIG. 28A shows the gRNA sequences used in the screen (SEQ IDNOs. 98-126). FIG. 28B shows the expression of TIM-3 in T cellsdisrupted by each TIM-3 gRNA. FIG. 28C shows the efficiency of TIM-3knockout/knockdown for each gRNA tested. FIG. 28D shows the expressionof CD3, PD1, or TIM-3 of T cells with TRAC/TRBC/PD1/TIM-3 disrupted, andT cells with TRAC/TRBC/PD1 disrupted.

TIM-3 CRISPR disruption shows further efficacy (FIGS. 29A-29B). FIG. 29Ashows a graph of tumor size of mice challenged with A549-ESO tumor (n=5)and treated with: TRAC/TRBC disrupted T cells transduced with 8FNY-ESO-1 TCR (8F.DK), TRAC/TRBC/PD1 disrupted T cells transduced with 8FNY-ESO-1 TCR (8F.TK-PD1), TRAC/TRBC/TIM-3 disrupted T cells transducedwith 8F NY-ESO-1 TCR (8F.TK-Tim3), TRAC/TRBC/PD1/TIM-3 disrupted T cellstransduced with 8F NY-ESO-1 TCR (8F.QK), T cells transduced with 8FNY-ESO-1 TCR (8F.TCR), TRAC/TRBC disrupted T cells transduced with 8FNY-ESO-1 TCR and PD1-CD28 switch receptor (8F.DKS), or TRAC/TRBC/TIM-3disrupted T cells transduced with 8F NY-ESO-1 TCR and PD1-CD28 switchreceptor (8F.TKS). No TD: non-transduced T cells. FIG. 29B showsbioluminescence imaging (BLI) of the treated mice.

Improved anti-tumor function of NY-ESO-1 TCR transduced T cells withPD1-CD28 switch receptor was found to be associated with significantgene expression profile changes (FIGS. 30A-30C). FIG. 30A shows thenumber of tumor infiltrating lymphocytes (TILs) isolated from mice withdifferent treatments (n=3). Cont.: control; DK: treatment with TRAC/TRBCdisrupted T cells transduced with 8F TCR; TK: treatment withTRAC/TRBC/PD1 disrupted T cells transduced with 8F TCR; DKS: TRAC/TRBCdisrupted T cells transduced with 8F TCR and PD1-CD28 switch receptor.FIG. 30B shows the expression of CD137 of the TILs stimulated byA549-ESO tumor line. FIG. 30C shows the IFN-gamma secretion of the TILsstimulated with A549-ESO tumor line.

RNA-seq data showed massive gene expression profile changes in T cellactivation, CD28 costimulatory signaling for PD1-CD28 NY-ESO-1 T cellswith TRAC/TRBC double disruption (FIGS. 31A-31F). FIG. 31A provides anoverview of the differentially expressed genes obtained from RNA-seqresults. FIG. 31B shows the Gene Set Enrichment Analysis comparingTRAC/TRBC disrupted T cells transduced with 8F and PD1-CD28 switch (DKS)and TRAC/TRBC disrupted T cells transduced with 8F (DK). The Gene SetEnrichment Analysis revealed 1790 enriched genes involved in 362 genesets. FIG. 31C shows that immune responses, T cell activation, nucleicacid metabolic processes, gene synthesis/expression, and cell cycle areamong the top 20 ranked pathways. FIG. 31D shows Gene Set EnrichmentAnalysis of DK versus DKS for immune system process pathway. FIG. 31Eshows a heat map for CD28 pathway associated genes. FIG. 31F shows aheat map for cytokine receptors that associate with T cell function. TK:TRAC/TRBC/PD1 disrupted T cells transduced with 8F TCR.

FIGS. 31G and 31H show that tumor-infiltrating lymphocytes (TILs)expressing NY-ESO-1 TCR and PD1-CD28 switch exhibit a uniquedistribution of gene expression. FIG. 31G shows the distribution ofvarious TILs as indicated in an A549 tumor slice. As shown, NY-ESO-1TILs expressing PD1-CD28 switch has higher distribution on the rightside of the tumor slice. The bottom image is an overlay of all fourdistributions. DK: NY-ESO-1 TCR T cells with TRAC/TRBC disrupted;TK-PD1: NY-ESO-1 TCR T cells with TRAC/TRBC/PDCD1 disrupted; DKS:NY-ESO-1 TCR T cells with TRAC/TRBC disrupted and expressing PD1-CD28switch; NTD: non-transduced. FIG. 31H shows Gene Set Enrichment Analysisof DK versus DKS for regulation of cell activation, immune systemprocess, regulation of cell-cell adhesion, T cell receptor signalingpathway, receptor activity, and signal transducer activity as indicated.

Example 3: PD1-CD28 Switch Receptor Boosts NY-ESO-1 TCR T Cell FunctionThrough Evading Suppression from Tumor Microenvironment

Transforming growth factor β (TGFβ) and adenosine are knownimmunosuppressive signals that reside within the tumor microenvironment.As shown in FIGS. 32A-32B, T cells transduced with NY-ESO-1 TCR (8F) andPD1-CD28 switch receptor exhibit increased resistance to TGFβ andadenosine, as compared to non-PD1-CD28 switch receptor transduced Tcells. FIG. 32A shows the resistance to TGFβ and resistance to adenosineinhibition of NY-ESO-1 TCR T cells. FIG. 32B shows the expression ofCD107a in different T cells stimulated with A549-ESO tumor line, in thepresence of either TGFβ or Adenosine. 8F DK: TRAC/TRBC disrupted T cellstransduced with 8F TCR; 8F.TK.PD1: TRAC/TRBC/PD1 disrupted T cellstransduced with 8F TCR; 8F.TK.Tim3: TRAC/TRBC/TIM-3 disrupted T cellstransduced with 8F TCR; 8F DKS: TRAC/TRBC disrupted T cells transducedwith 8F TCR and PD1-CD28 switch receptor; 8F TKS.Tim3: TRAC/TRBC/TIM-3disrupted T cells transduced with 8F TCR and PD1-CD28 switch receptor.

Regulatory T cells (Tregs) are known to inhibit the function of T cells.As shown in FIGS. 33A-33B, 8F DKS cells demonstrate enhanced resistanceto Tregs, as compared to 8F DK and 8F.TK.PD1 cells. FIG. 33A shows theresistance to Treg inhibition of NY-ESO-1 TCR T cells. FIG. 33B showscarboxyfluorescein succinimidyl ester (CFSE) dilution of different Tcells stimulated with A549-ESO tumor line, in the presence of differentamount of Tregs.

The PD1-CD28 switch receptor was also found to confer NY-ESO-1 TCR Tcells resistance to hypoxia inhibition. FIG. 33C demonstrates enhancedresistance of various 8F T cells (NY-ESO-1 TCR T cells) as indicated tohypoxia inhibition, by measuring IFNγ production in the various media asindicated. 8F.DK indicates 8F NY-ESO-1 TCR T cells with disrupted TRACand TRBC; 8F.TK indicates 8F NY-ESO-1 TCR T cells with disrupted TRAC,TRBC, and PDCD1; 8F.DKS indicates 8F NY-ESO-1 TCR T cells with disruptedTRAC and TRBC, additionally expressing a switch receptor.

Example 4: TGFβR-IL12R Switch Receptor

A TGFβR-IL12R switch receptor also further improves NY-ESO-1 TCRtransduced T cell function. A TGFβR-IL12R switch receptor was found toboost T cell function (FIGS. 34A-34B). FIG. 34A shows a schematic of theTGFβR-IL12R switch receptor. FIG. 34B shows the level of cytokineproduction of different NY-ESO-1 TCR transferred T cells that wereco-transferred with TGFβR-IL12R switch receptor, or dominant negativeTGFβR(DN), in the presence or absence of TGFβI.

Example 5: High Affinity PD1 Switch Receptor Enhances NY-ESO-1 TCR TCell Function

Several switch receptors were generated using methods known in the art,and using sequences described elsewhere herein. The high affinity PD1switch receptor, PD1^(A132L)-41BB, comprises a variant PD1 extracellulardomain having an A132L substitution relative to the full length aminoacid sequence of PD1, a CD8alpha transmembrane domain, and a 4-1BBcytoplasmic domain. The PD1 A132L substitution increases its affinityfor PD-L1. See, e.g., Zhang et al. Immunity 2004, 20:337-347. PD1-41BB,TIM3-CD28 and PD1^(A132L)-CD28 switch receptors were also generatedusing methods known in the art, and using sequences described elsewhereherein.

5×10⁶ tumor (A549ESO) cells in Matrigel™ were injected (s.c.) on day 0.On day 8, 10×10⁶ 8F-VP8 positive T cells were injected intravenously.Tumor growth was monitored by bioluminescence imaging (BLI) weekly.NY-ESO-1 TCR (8F) positive T cells had the following phenotypes:NY-ESO-1 TCR (8F) only; NY-ESO-1 TCR (8F) and PD1.CD28 switch(PD1-CD28); NY-ESO-1 TCR (8F) and PD1*.CD28 switch (PD1^(A132L)-CD28);NY-ESO-1 TCR (8F) and PD1.BB switch (PD1-41BB); or NY-ESO-1 TCR (8F) andPD1*.BB switch (PD1^(A132L)-411BE). Untransduced T cells (UTD) were usedas a control. In FIG. 35A, BLI of mice injected with T cells asindicated shows that the high affinity PD1 switches (i.e., PD1*.CD28 andPD1*.BB) enhance the anti-tumor activity of NY-ESO-1 redirected T cells.FIG. 35B shows the quantification of average radiance for the indicatedgroups. FIG. 35C shows a plot of tumor size measured weeklypost-injection with T cells for the various groups as indicated. UTD:untransduced; CD28: NY-ESO-1 TCR (8F) and PD1.CD28 switch (PD1-CD28);CD28 #: NY-ESO-1 TCR (8F) and PD1*.CD28 switch (PD1^(A132L)-CD28); BB:NY-ESO-1 TCR (8F) and PD1.1B switch (PD1-41BB); or BB #: NY-ESO-1 TCR(8F) and PD1*.BB switch (PD1^(A132L)-41BB).

In FIG. 36A, BLI of mice injected with T cells as indicated showed thatthe high affinity PD1 switches (i.e., PD1*.CD28 and PD1*.BB) enhancedthe anti-tumor activity of NY-ESO-1 redirected T cells. FIG. 36B showsthe quantification of average radiance for the indicated groups. FIG.36C shows a plot of tumor size measured weekly post-injection with Tcells for the various groups as indicated. The various groups are asfollows: from top to bottom, A: untransduced, TRAC/TRBC disrupted Tcells (UTD DKO); B: NY-ESO-1 TCR (8F), TRAC/TRBC disrupted T cells (8FDKO); C: NY-ESO-1 TCR (8F), TRAC/TRBC/PDCD1/TIM3 disrupted T cells (8FDKO+PD1 & Tim3 KO); D: NY-ESO-1 TCR (8F), TIM3-CD28 switch, TRAC/TRBCdisrupted T cells (Tim3CD28.8F DKO); E: NY-ESO-1 TCR (8F), TIM3-CD28switch, TRAC/TRBC/PDCD1 disrupted T cells (Tim3CD28.8F DKO+PD1 KO); F:NY-ESO-1 TCR (8F), PD1^(A132L)-41BB switch, TRAC/TRBC disrupted T cells(PD1*BB.8F DKO); G: NY-ESO-1 TCR (8F), PD1^(A132L)-41BB switch,TRAC/TRBC/TIM3 disrupted T cells (PD1*BB.8F DKO+ Tim3 KO); H: NY-ESO-1TCR (8F), PD1^(A132L)-41BB switch, TIM3-CD28 switch, TRAC/TRBC disruptedT cells (PD1*BB.Tim3CD28.8F DKO); and I: NY-ESO-1 TCR (8F), PD1-CD28switch, TRAC/TRBC/TIM3 disrupted T cells (PD1CD28.8F DKO+ Tim3 KO). FIG.36D shows BLI of mice injected with T cells as indicated, demonstratingthat high affinity PD1 switch receptors enhanced 8F NY-ESO-1 TCRre-directed anti-tumor activity. FIG. 36E shows a plot of tumor sizemeasured at various time points as indicated post-injection with T cellsfor the various groups as indicated. In FIGS. 36D and 36E, UTD:untransduced T cells; PD1.CD28.8F (CD28 in FIG. 36E): 8F NY-ESO-1 TCR Tcells with PD1-CD28 switch; PD1*.CD28.8F (CD28 #in FIG. 36E): 8FNY-ESO-1 TCR T cells with PD1^(A132L) switch; PD1.EE.8F (BB in FIG.36E): 8F NY-ESO-1 TCR T cells with PD1-41BB switch; PD1*.BB.8F (BB #inFIG. 36E): 8F NY-ESO-1 TCR T cells with PD1^(A132L)-41 BB switch; 8F: 8FNY-ESO-1 TCR T cells.

1.-24. (canceled)
 25. A method for generating a modified T cellcomprising: a) introducing into a T cell a first nucleic acid comprisinga nucleic acid sequence encoding an exogenous T cell receptor (TCR)having affinity for NY-ESO-1 on a target cell; and b) introducing intothe T cell one or more nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from the groupconsisting of TCR alpha chain, and TCR beta chain.
 26. The method ofclaim 25, wherein the one or more nucleic acids capable ofdownregulating gene expression comprises a CRISPR system comprising aCas9/gRNA ribonucleoprotein (RNP) complex.
 27. The method of claim 25,wherein the first nucleic acid further comprises a nucleic acid sequenceencoding a switch receptor.
 28. The method of claim 27, wherein thenucleic acid sequence encoding the switch receptor comprises the nucleicacid sequence set forth in SEQ ID NOs: 15, 135, 137, or
 139. 29. Themethod of 25, further comprising: c) introducing into the T cell anucleic acid capable of downregulating gene expression of an endogenousPD1 coding sequence.
 30. The method of claim 29, wherein the one or morenucleic acids capable of downregulating gene expression comprises aCRISPR system comprising a Cas9/gRNA ribonucleoprotein (RNP) complex.31. The method of claim 25, wherein the exogenous TCR comprises: (i) aTCR alpha chain comprising the amino acid sequence set forth in SEQ IDNO:5; and/or (ii) a TCR beta chain comprising the amino acid sequenceset forth in SEQ ID NO:
 12. 32. The method of claim 25, wherein: (i) theendogenous TCR alpha chain gene comprises the nucleic acid sequence setforth in SEQ ID NO:128; and/or (ii) the endogenous TCR beta chain genecomprises the nucleic acid sequence set forth in SEQ ID NO:129.
 33. Themethod of claim 29, wherein the endogenous PD1 coding sequencecomprising the nucleic acid sequence set forth in SEQ ID NO:130.
 34. Themethod of claim 27, wherein the switch receptor comprises: a firstdomain, wherein the first domain is derived from a first polypeptidethat is associated with a negative signal; a second domain comprising aswitch receptor transmembrane domain; and a third domain, wherein thethird domain is derived from a second polypeptide that is associatedwith a positive signal.
 35. The method of claim 34, wherein the firstpolypeptide that is associated with a negative signal is PD-1.
 36. Themethod of claim 34, wherein the first polypeptide that is associatedwith a negative signal is a variant of PD-1 having an alanine-to-leucinesubstitution at amino acid position 132 relative to the wild-type PD-1amino acid sequence.
 37. The method of claim 34, wherein the thirdpolypeptide that is associated with a positive signal is CD28 or 4-1BB.38. The method of claim 34, wherein the switch receptor comprises: afirst domain comprising at least a portion of the extracellular domainof PD1; a second domain comprising a switch receptor transmembranedomain comprising at least a portion of the transmembrane domain ofCD28; and a third domain comprising at least a portion of theintracellular domain of CD28.
 39. The method of claim 34, wherein theswitch receptor comprises: a first domain comprising at least a portionof the extracellular domain of PD-1; a second domain comprising a switchreceptor transmembrane domain comprising at least a portion of thetransmembrane domain of CD8alpha; and a third domain comprising at leasta portion of the intracellular domain of 4-1BB.
 40. The method of claim34, wherein the switch receptor comprises: a first domain comprising atleast a portion of the extracellular domain of PD-1, wherein the PD-1 isa variant having an alanine-to-leucine substitution at amino acidposition 132 relative to the wild-type PD-1 amino acid sequence; asecond domain comprising a switch receptor transmembrane domaincomprising at least a portion of the transmembrane domain of CD28; and athird domain comprising at least a portion of the intracellular domainof CD28.
 41. The method of claim 34, wherein the switch receptorcomprises: a first domain comprising at least a portion of theextracellular domain of PD-1, wherein the PD-1 is a variant having analanine-to-leucine substitution at amino acid position 132 relative tothe wild-type PD-1 amino acid sequence; a second domain comprising aswitch receptor transmembrane domain comprising at least a portion ofthe transmembrane domain of CD8alpha; and a third domain comprising atleast a portion of the intracellular domain of 4-1BB.
 42. The method ofclaim 34, wherein the switch receptor comprises the amino acid sequenceset forth in any one of SEQ ID NOs: 14, 134, 136, or
 138. 43. The methodof claim 25, wherein T cell is an autologous T cell derived from ahuman.